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Sample records for hsp70 stress gene

  1. Genome-wide analysis of the Hsp70 family genes in pepper (Capsicum annuum L.) and functional identification of CaHsp70-2 involvement in heat stress.

    Science.gov (United States)

    Guo, Meng; Liu, Jin-Hong; Ma, Xiao; Zhai, Yu-Fei; Gong, Zhen-Hui; Lu, Ming-Hui

    2016-11-01

    Hsp70s function as molecular chaperones and are encoded by a multi-gene family whose members play a crucial role in plant response to stress conditions, and in plant growth and development. Pepper (Capsicum annuum L.) is an important vegetable crop whose genome has been sequenced. Nonetheless, no overall analysis of the Hsp70 gene family is reported in this crop plant to date. To assess the functionality of Capsicum annuum Hsp70 (CaHsp70) genes, pepper genome database was analyzed in this research. A total of 21 CaHsp70 genes were identified and their characteristics were also described. The promoter and transcript expression analysis revealed that CaHsp70s were involved in pepper growth and development, and heat stress response. Ectopic expression of a cytosolic gene, CaHsp70-2, regulated expression of stress-related genes and conferred increased thermotolerance in transgenic Arabidopsis. Taken together, our results provide the basis for further studied to dissect CaHsp70s' function in response to heat stress as well as other environmental stresses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

    Science.gov (United States)

    Hangzo, Hnunlalliani; Banerjee, Bodhisattwa; Saha, Shrabani; Saha, Nirmalendu

    2017-02-01

    The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90β) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH 4 Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90β genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

  3. Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

    Science.gov (United States)

    Cubano, L. A.; Lewis, M. L.

    2001-01-01

    Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

  4. Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

    Science.gov (United States)

    Yan, Jie; Liang, Xiao; Zhang, Yin; Li, Yang; Cao, Xiaojuan; Gao, Jian

    2017-07-01

    Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Identification and in silico analysis of the Citrus HSP70 molecular chaperone gene family

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    Luciano G. Fietto

    2007-01-01

    Full Text Available The completion of the genome sequencing of the Arabidopsis thaliana model system provided a powerful molecular tool for comparative analysis of gene families present in the genome of economically relevant plant species. In this investigation, we used the sequences of the Arabidopsis Hsp70 gene family to identify and annotate the Citrus Hsp70 genes represented in the CitEST database. Based on sequence comparison analysis, we identified 18 clusters that were further divided into 5 subgroups encoding four mitochondrial mtHsp70s, three plastid csHsp70s, one ER luminal Hsp70 BiP, two HSP110/SSE-related proteins and eight cytosolic Hsp/Hsc70s. We also analyzed the expression profile by digital Northern of each Hsp70 transcript in different organs and in response to stress conditions. The EST database revealed a distinct population distribution of Hsp70 ESTs among isoforms and across the organs surveyed. The Hsp70-5 isoform was highly expressed in seeds, whereas BiP, mitochondrial and plastid HSp70 mRNAs displayed a similar expression profile in the organs analyzed, and were predominantly represented in flowers. Distinct Hsp70 mRNAs were also differentially expressed during Xylella infection and Citrus tristeza viral infection as well as during water deficit. This in silico study sets the groundwork for future investigations to fully characterize functionally the Citrus Hsp70 family and underscores the relevance of Hsp70s in response to abiotic and biotic stresses in Citrus.

  6. Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

    Science.gov (United States)

    Khalouei, Sam; Chow, Ari M; Brown, Ian R

    2014-05-01

    The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

  7. Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress.

    Science.gov (United States)

    Long, Ling-Li; Han, Ying-Li; Sheng, Zhang; Du, Chen; Wang, You-Fa; Zhu, Jun-Quan

    2015-09-01

    The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. thermal stress and Hsp70 as selective agents

    Indian Academy of Sciences (India)

    Madhu Sudhan

    starred in an exhaustive list of studies linking its expression to environmental ... genetic stress linked to Hsp70: polyglutamine expansion .... Twenty-five D. melanogaster genes conserved in all twelve Drosophila species (fourth column) possessing conserved ...... like growth factor-1, and vertebral bone mass in men; J. Clin.

  9. PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS

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    Mosenson, Jeffrey A.; Flood, Kelsey; Klarquist, Jared; Eby, Jonathan M.; Koshoffer, Amy; Boissy, Raymond E.; Overbeck, Andreas; C.Tung, Rebecca; Poole, I. Caroline Le

    2014-01-01

    SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes. PMID:24354861

  10. Rice sHsp genes: genomic organization and expression profiling under stress and development

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    Grover Anil

    2009-08-01

    Full Text Available Abstract Background Heat shock proteins (Hsps constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20, Hsp20 or small Hsps (sHsps are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes. Results We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed. Conclusion We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed

  11. UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

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    Fulgentini, Lorenzo; Passini, Valerio; Colombetti, Giuliano; Miceli, Cristina; La Terza, Antonietta; Marangoni, Roberto

    2015-08-01

    The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

  12. Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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    Michael Roth

    2009-01-01

    Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

  13. Phenotypic Plasticity of HSP70s Gene Expression during Diapause: Signs of Evolutionary Responses to Cold Stress among Soybean Pod Borer Populations (Leguminivora glycinivorella) in Northeast of China

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    Han, Lanlan; Fan, Dong; Zhao, Kuijun

    2014-01-01

    The soybean pod borer (Leguminivora glycinivorella Matsumura) successfully survives the winter because of its high expression of 70-kDa heat shock proteins (HSP70s) during its overwintering diapause. The amount of HSP70s is different under different environmental stresses. In this study, inducible heat shock protein 70 and its constitutive heat shock cognate 70 were cloned by RT-PCR and RACE. These genes were named Lg-hsp70 and Lg-hsc70, respectively. Gene transcription and protein expression after cold stress treatment (5°C to −5°C) were analyzed by western blotting and by qRT-PCR for four populations that were sampled in the northeast region of China, including Shenyang, Gongzhuling, Harbin and Heihe, when the soybean pod borer was in diapause. As the cold shock temperature decreased, the levels of Lg-HSP70s were significantly up-regulated. The amount of cold-induced Lg-HSP70s was highest in the southernmost population (Shenyang, 41°50′N) and lowest in the northernmost population (Heihe, 50°22′N). These results support the hypothesis that the soybean pod borer in the northeast region of China displays phenotypic plasticity, and the accumulation of Lg-HSP70s is a strategy for overcoming environmental stress. These results also suggest that the induction of HSP70 synthesis, which is a complex physiological adaptation, can evolve quickly and inherit stability. PMID:25330365

  14. 70 kD stress protein (Hsp70) analysis in living shallow-water benthic foraminifera

    Digital Repository Service at National Institute of Oceanography (India)

    Heinz, P.; Marten, R.A; Linshy, V.N.; Haap, T.; Geslin, E.; Kohler, H-R.

    Hsp70 is a phylogenetically highly conserved protein family present in all eukaryotic organisms tested so far. Its synthesis is induced by proteotoxic stress. The detection of Hsp70 in foraminifera is presented here. We introduce a standard...

  15. Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis.

    Science.gov (United States)

    Zhu, Yongfei; Ren, Chuanlu; Wan, Xuying; Zhu, Yuping; Zhu, Jiangbo; Zhou, Hongyuan; Zhang, Tianbao

    2013-11-01

    Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.

  16. Association of eNOS and HSP70 gene polymorphisms with glaucoma in Pakistani cohorts.

    NARCIS (Netherlands)

    Ayub, H.; Khan, M.I.; Micheal, S.; Akhtar, F.; Ajmal, M.; Shafique, S.; Ali, S.H.; Hollander, A.I. den; Ahmed, A.; Qamar, R.

    2010-01-01

    PURPOSE: To investigate the involvement of stress-regulating genes, endothelial nitric oxide synthase (eNOS) and heat shock protein 70 (HSP70) with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG). METHODS: POAG and PCAG patients recruited from different areas of Pakistan

  17. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail: ionan.marigomez@ehu.es

    2014-04-01

    Highlights: • Thermal stress and Cd caused lysosomal enlargement and membrane destabilisation. • hex, gusb and ctsl but not hsp70 were up-regulated at elevated temperature but down-regulated by Cd. • Thermal stress influenced lysosomal responses to Cd exposure. • The presence of Cd jeopardised responsiveness against thermal stress. - Abstract: In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24 h at 18 °C and 26 °C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18 °C and 26 °C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution

  18. Association of HSP70-2 Gene 1267A/G Polymorphism With Cataract Incidence Among Guilan Population

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    Zivar Salehi

    2017-01-01

    Full Text Available Background: Cataract is a visible opacity of the eye lens and it is the main reason of reversible blindness in the world. Oxidative stress is known as a major factor in cataract formation HSP70-2 protein is a molecular chaperone which is essential for cell survival in stress conditions. HSP70-2 gene is located in the human leukocyte antigen class ΙΙΙ region. This gene encodes an inducible protein. One of the common polymorphism of HSP70-2 is 1267A/G which is located in coding region. The aim of this study was to analysis of 1267A/G polymorphism of hsp70-2 gene in cataract patients. Material and Methods: This case-control study included 118 cataract patients and 122 healthy people as a control groups. Genomic DNA was extracted from peripheral blood leukocytes and genotyping determination was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. Statistical analysis was performed by using the MedCalc software (Version 12.1. Results: The frequency of G allele was significantly higher in patients than the controls, (0.36 and 0.24, respectively. A higher frequency of the GG genotype of the HSP70-2 1267A/G polymorphism was found in the patients compared with controls (21.19% and 8.20%, respectively. The patients carrying the GG genotype were 3.2-fold at a higher risk of cataract compared with AA genotype (P=0.005. Conclusion: The finding of this study suggested that the HSP70-2 1267A/G may affect the susceptibility to cataract in the studied population. Anyway the randomized multicenter studies with greater sample size still need to confirmed our results.

  19. Expression analysis of NOS family and HSP genes during thermal stress in goat ( Capra hircus)

    Science.gov (United States)

    Yadav, Vijay Pratap; Dangi, Satyaveer Singh; Chouhan, Vikrant Singh; Gupta, Mahesh; Dangi, Saroj K.; Singh, Gyanendra; Maurya, Vijay Prakash; Kumar, Puneet; Sarkar, Mihir

    2016-03-01

    Approximately 50 genes other than heat shock protein (HSP) expression changes during thermal stress. These genes like nitric oxide synthase (NOS) need proper attention and investigation to find out their possible role in the adaptation to thermal stress in animals. So, the present study was undertaken to demonstrate the expressions of inducible form type II NOS (iNOS), endothelial type III NOS (eNOS), constitutively expressed enzyme NOS (cNOS), HSP70, and HSP90 in peripheral blood mononuclear cells (PBMCs) during different seasons in Barbari goats. Real-time polymerase chain reaction, western blot, and immunocytochemistry were applied to investigate messenger RNA (mRNA) expression, protein expression, and immunolocalization of examined factors. The mRNA and protein expressions of iNOS, eNOS, cNOS, HSP70, and HSP90 were significantly higher ( P goats.

  20. Heat shock proteins (Hsp 70) response is not systematic to cell stress

    International Nuclear Information System (INIS)

    Hassen, Wafa; Ayed-Boussema, Imen; Bouslimi, Amel; Bacha, Hassen

    2007-01-01

    Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [ 3 H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress

  1. Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress.

    Science.gov (United States)

    Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Yusuff, Oladosu A; Ismail, Mohd R; Miah, Gous

    2017-04-01

    Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.

  2. Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

    Science.gov (United States)

    Guo, Meng; Zhai, Yu-Fei; Lu, Jin-Ping; Chai, Lin; Chai, Wei-Guo; Gong, Zhen-Hui; Lu, Ming-Hui

    2014-01-01

    Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl2, H2O2 and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca2+, H2O2 and Put. PMID:25356507

  3. Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment

    International Nuclear Information System (INIS)

    Stangl, Stefan; Themelis, George; Friedrich, Lars; Ntziachristos, Vasilis; Sarantopoulos, Athanasios; Molls, Michael; Skerra, Arne; Multhoff, Gabriele

    2011-01-01

    Background and purpose: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. Materials and methods: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. Results: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 deg. C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 deg. C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. Conclusions: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

  4. EFFECT OF STRESS ON THE PERCENT BODY WEIGHT CHANGE AND MRNA EXPRESSION OF IGF-1, SURVIVINE AND HSP-70 GENE IN THE HIERARCHIAL FOLLICLES OF JAPANESE QUAIL

    Directory of Open Access Journals (Sweden)

    N Shit

    2014-12-01

    Full Text Available The present study was carried out to explore the effect of stress on body weight and the mRNA expression of IGF-1, Survivine and HSP-70 gene in the hierarchial follicles of Japanese quail. A total 24 birds (10 weeks were taken and stress was induced by immobilization daily for 2hrs (between 9.00 - 11.00 AM throughout the study (10 days. Four birds were sacrificed on 1, 2, 4, 6, 8 and 10 days of the treatment. Hierarchial follicles (F1, F2 & F3 were aseptically collected to quantify the expression of IGF-1, Survivine and HSP-70 gene using real-time PCR technique. The percent body weight reduction increased and reached highest (21.30% on 10th day. The fold expression of IGF-1 gene was significantly ((P=0.05 down regulated in advance to the time of experiment. However, the fold expression of survivine gene was significantly (P=0.05 up regulated and the intensity was highest (17 fold in F-3 follicle on 4th day of experiment. No significant change in the mRNA expression of HSP-70 gene was evident in this study. From this study it may be concluded that stress brings physio-molecular change through HPA activation, which not only causes tissue regression also modifies the cellular mechanism.

  5. B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

    Science.gov (United States)

    Navarro-Domínguez, Beatriz; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2016-10-01

    As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes.

  6. Characterization of six small HSP genes from Chironomus riparius (Diptera, Chironomidae): Differential expression under conditions of normal growth and heat-induced stress.

    Science.gov (United States)

    Martín-Folgar, Raquel; de la Fuente, Mercedes; Morcillo, Gloria; Martínez-Guitarte, José-Luis

    2015-10-01

    Small heat shock proteins (sHSPs) comprise the most numerous, structurally diverse, and functionally uncharacterized family of heat shock proteins. Several Hsp genes (Hsp 90, 70, 40, and 27) from the insect Chironomus riparius are widely used in aquatic toxicology as biomarkers for environmental toxins. Here, we conducted a comparative study and characterized secondary structure of the six newly identified sHsp genes Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, and Hsp34. A characteristic α-crystallin domain is predicted in all the new proteins. Phylogenetic analysis suggests a strong relation to other sHSPs from insects and interesting evidence regarding evolutionary origin and duplication events. Comparative analysis of transcription profiles for Hsp27, Hsp70, and the six newly identified genes revealed that Hsp17, Hsp21, and Hsp22 are constitutively expressed under normal conditions, while under two different heat shock conditions these genes are either not activated or are even repressed (Hsp22). In contrast, Hsp23, Hsp24, and Hsp34 are significantly activated along with Hsp27 and Hsp70 during heat stress. These results strongly suggest functional differentiation within the small HSP subfamily and provide new data to help understand the coping mechanisms induced by stressful environmental stimuli. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

    Science.gov (United States)

    Bharati, Jaya; Dangi, S S; Chouhan, V S; Mishra, S R; Bharti, M K; Verma, V; Shankar, O; Yadav, V P; Das, K; Paul, A; Bag, S; Maurya, V P; Singh, G; Kumar, P; Sarkar, M

    2017-06-01

    Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

  8. Concerted and nonconcerted evolution of the Hsp70 gene superfamily in two sibling species of nematodes.

    Science.gov (United States)

    Nikolaidis, Nikolas; Nei, Masatoshi

    2004-03-01

    We have identified the Hsp70 gene superfamily of the nematode Caenorhabditis briggsae and investigated the evolution of these genes in comparison with Hsp70 genes from C. elegans, Drosophila, and yeast. The Hsp70 genes are classified into three monophyletic groups according to their subcellular localization, namely, cytoplasm (CYT), endoplasmic reticulum (ER), and mitochondria (MT). The Hsp110 genes can be classified into the polyphyletic CYT group and the monophyletic ER group. The different Hsp70 and Hsp110 groups appeared to evolve following the model of divergent evolution. This model can also explain the evolution of the ER and MT genes. On the other hand, the CYT genes are divided into heat-inducible and constitutively expressed genes. The constitutively expressed genes have evolved more or less following the birth-and-death process, and the rates of gene birth and gene death are different between the two nematode species. By contrast, some heat-inducible genes show an intraspecies phylogenetic clustering. This suggests that they are subject to sequence homogenization resulting from gene conversion-like events. In addition, the heat-inducible genes show high levels of sequence conservation in both intra-species and inter-species comparisons, and in most cases, amino acid sequence similarity is higher than nucleotide sequence similarity. This indicates that purifying selection also plays an important role in maintaining high sequence similarity among paralogous Hsp70 genes. Therefore, we suggest that the CYT heat-inducible genes have been subjected to a combination of purifying selection, birth-and-death process, and gene conversion-like events.

  9. Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

    Science.gov (United States)

    Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

    2018-03-01

    The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

  10. High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong, E-mail: zhangqzdr@126.com

    2016-06-10

    HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl{sub 2} stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. -- Highlights: •HMG protein ChDSP1 shows affinity to ChHSP70 promoter in Crassostrea hongkongensis. •ChDSP1 negatively regulates ChHSP70 transcription. •ChHSP70 and ChDSP1 transcriptions were coordinately induced by thermal/Cd stress. •ChDSP1 may contribute to the recovery of the induced ChHSP70 to its original state. •This is the first report regarding negative regulator of HSP70 transcription.

  11. Investigating hsp Gene Expression in Liver of Channa striatus under Heat Stress for Understanding the Upper Thermal Acclimation

    Directory of Open Access Journals (Sweden)

    Gopal Krishna Purohit

    2014-01-01

    Full Text Available Changes in hsp gene expression profiles in murrel Channa striatus experimentally exposed to temperature stress (36°C for 4, 15, and 30 days were investigated; fish collected from aquaculture ponds and maintained in laboratory at the pond temperature (25 ± 1°C served as control. Channa collected from a hot spring runoff (36°C was included in the study to examine the hsp profiles beyond 30 days of exposure. Gene expression analyses of a battery of hsps in liver tissues were carried out by quantitative RT-PCR and protein expressions were analyzed by immunoblotting. hsps could be grouped into three clusters based on similarity in response to heat stress: hsp70, hsp78, and hsp60, whose transcript level continued to increase with duration of exposure; hsp90 and hsp110 that increased to a much higher level and then decreased; hsp27 and hsp47 that did not significantly vary as compared to control. The results suggest that Hsp70, Hsp78, and Hsp60 are involved in thermal acclimation and long term survival at high temperature. Fish living in the hot spring runoff appears to continuously express hsps that can be approximated by long term induction of hsps in farmed fish if temperature of their environment is raised to 36°C.

  12. Increased HSF activation in muscles with a high constitutive Hsp70 expression

    OpenAIRE

    Locke, Marius; Tanguay, Robert M.

    1996-01-01

    Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5′ region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of ...

  13. Effects of chronic thermal stress on growth performance, carcass traits, antioxidant indices and the expression of HSP70, growth hormone and superoxide dismutase genes in two broiler strains.

    Science.gov (United States)

    Roushdy, Elshimaa M; Zaglool, Asmaa W; El-Tarabany, Mahmoud S

    2018-05-01

    The objective was to investigate the effects of genetic type and the duration of chronic thermal stress (36 °C) on the growing efficiency, carcass traits, antioxidant status, and the expression of liver heat shock protein 70 (HSP70), growth hormone (GH) and superoxide dismutase (SOD) genes. Two hundred and seventy one-day-old chicks (135 male chicks of each breed; Ross 308 and Cobb 500) were used in this work. On the 21st day of age, birds were allocated randomly into 3 equal groups till the 42 days of age (CON:raised in a thermoneutral condition; HS 1 and HS 2 groups were subjected to 4 and 6 h of daily thermal stress, respectively). Regardless of genetic type, thermal stress decreased the dressing percentage in broilers when compared with the thermoneutral conditions (p = 0.039). In both broiler strains, thermal stress for 6 h (HS 2 ) increased the heterophil to lymphocyte ratio (p = 0.036) and the serum albumin, cholesterol and triglyceride levels (p = 0.023, 0.012 and 0.005, respectively) compared with the thermoneutral group. Under the thermonuteral and heat stress conditions, the Ross broiler chickens showed a significant lower serum triiodothyronine level compared with the Cobb boilers (p = 0.042). It is interesting to note that the expression of HSP70 in the liver of heat-stressed Ross broilers, either 4 or 6 h, was significantly (p = 0.002) higher than that reported in the heat-stressed Cobb broilers. In both broiler strains, the thermal stress for 6 h up-regulate the expression of SOD gene (p = 0.001), but down-regulate the expression of GH gene (p = 0.021) when compared with the CON group. In conclusion, chronic thermal stress down-regulate the mRNA expression of liver GH, concomitantly with an increase in the expression of HSP70 and SOD genes in both broiler strains. This could be useful in the identification of molecular genetic markers to assist in selecting broilers that are more tolerant to heat stress

  14. Role of membrane Hsp70 in radiation sensitivity of tumor cells

    International Nuclear Information System (INIS)

    Murakami, Naoya; Kühnel, Annett; Schmid, Thomas E.; Ilicic, Katarina; Stangl, Stefan; Braun, Isabella S.; Gehrmann, Mathias; Molls, Michael; Itami, Jun; Multhoff, Gabriele

    2015-01-01

    The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in the cytosol and integrated in the plasma membrane of tumor cells via lipid anchorage. Following stress such as non-lethal irradiation Hsp70 synthesis is up-regulated. Intracellular located Hsp70 is known to exert cytoprotective properties, however, less is known about membrane (m)Hsp70. Herein, we investigate the role of mHsp70 in the sensitivity towards irradiation in tumor sublines that differ in their cytosolic and/or mHsp70 levels. The isogenic human colon carcinoma sublines CX + with stable high and CX − with stable low expression of mHsp70 were generated by fluorescence activated cell sorting, the mouse mammary carcinoma sublines 4 T1 (4 T1 ctrl) and Hsp70 knock-down (4 T1 Hsp70 KD) were produced using the CRISPR/Cas9 system, and the Hsp70 down-regulation in human lung carcinoma sublines H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD was achieved by small interfering (si)RNA against Heat shock factor 1 (HSF-1). Cytosolic and mHsp70 was quantified by Western blot analysis/ELISA and flow cytometry; double strand breaks (DSBs) and apoptosis were measured by flow cytometry using antibodies against γH2AX and real-time PCR (RT-PCR) using primers and antibodies directed against apoptosis related genes; and radiation sensitivity was determined using clonogenic cell surviving assays. CX + /CX − tumor cells exhibited similar cytosolic but differed significantly in their mHsp70 levels, 4 T1 ctrl/4 T1 Hsp70 KD cells showed significant differences in their cytosolic and mHsp70 levels and H1339 ctrl/H1339 HSF-1 KD and EPLC-272H ctrl/EPLC-272H HSF-1 KD lung carcinoma cell sublines had similar mHsp70 but significantly different cytosolic Hsp70 levels. γH2AX was significantly up-regulated in irradiated CX − and 4 T1 Hsp70 KD with low basal mHsp70 levels, but not in their mHsp70 high expressing counterparts, irrespectively of their cytosolic Hsp70 content. After

  15. Metallothionein and Hsp70 trade-off against one another in Daphnia magna cross-tolerance to cadmium and heat stress

    Energy Technology Data Exchange (ETDEWEB)

    Haap, Timo, E-mail: timo.haap@gmx.de; Schwarz, Simon; Köhler, Heinz-R.

    2016-01-15

    Highlights: • Cadmium acclimation of two Daphnia magna clones which differed in Cd sensitivity and Hsp70 levels. • Two distinct metal-handling strategies regarding Hsp70 and MT expression were observed. • High Hsp70 levels did not confer an increase in Cd and heat stress tolerance. • Our results indicate a trade-off between Hsp70 and MT. - Abstract: The association between the insensitivity of adapted ecotypes of invertebrates to environmental stress, such as heavy metal pollution, and overall low Hsp levels characterizing these organisms has been attracting attention in various studies. The present study seeks to induce and examine this phenomenon in Daphnia magna by multigenerational acclimation to cadmium in a controlled laboratory setting. In this experiment, interclonal variation was examined: two clones of D. magna that have previously been characterized to diverge regarding their cadmium resistance and levels of the stress protein Hsp70, were continuously exposed to a sublethal concentration of Cd over four generations to study the effects of acclimation on Hsp70, metallothionein (MT), reproduction and cross-tolerance to heat stress. The two clones differed in all the measured parameters in a characteristic way, clone T displaying Cd and heat resistance, lower Hsp70 levels and offspring numbers on the one hand and higher MT expression on the other hand, clone S the opposite for all these parameters. We observed only slight acclimation-induced changes in constitutive Hsp70 levels and reproductive output. The differences in MT expression between clones as well as between acclimated organisms and controls give evidence for MT accounting for the higher Cd tolerance of clone T. Overall high Hsp70 levels of clone S did not confer cross tolerance to heat stress, contrary to common expectations. Our results suggest a trade-off between the efforts to limit the proteotoxic symptoms of Cd toxicity by Hsp70 induction and those to sequester and detoxify Cd by

  16. Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period.

    Science.gov (United States)

    Rout, P K; Kaushik, R; Ramachandran, N

    2016-07-01

    It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36-89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at -70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5-2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5

  17. Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians☆

    Science.gov (United States)

    Arisue, Nobuko; Sánchez, Lidya B.; Weiss, Louis M.; Müller, Miklós; Hashimoto, Tetsuo

    2011-01-01

    Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past. PMID:11880223

  18. De novo characterisation of the greenlip abalone transcriptome (Haliotis laevigata) with a focus on the heat shock protein 70 (HSP70) family.

    Science.gov (United States)

    Shiel, Brett P; Hall, Nathan E; Cooke, Ira R; Robinson, Nicholas A; Strugnell, Jan M

    2015-02-01

    Abalone (Haliotis) are economically important molluscs for fisheries and aquaculture industries worldwide. Despite this, genomic resources for abalone and molluscs are still limited. Here we present a description and functional annotation of the greenlip abalone (Haliotis laevigata) transcriptome. We present a focused analysis on the heat shock protein 70 (HSP70) family of genes with putative functions affecting temperature stress and immunity. A total of ~38 million paired end Illumina reads were obtained, resulting in a Trinity assembly of 222,172 contigs with minimum length of 200 base pairs and maximum length of 33 kilobases. The 20,702 contigs were annotated with gene descriptions by BLAST. We created a program to maximise the number of functionally annotated genes, and over 10,000 contigs were assigned Gene ontologies (GO terms). By using CateGOrizer, immunity related GO terms for stressors such as heat, hypoxia, oxidative stress and wounding received the highest counts. Twenty-six contigs with homology to the HSP70 family of genes were identified. Ninety-one putative single-nucleotide polymorphisms were observed in the abalone HSP70 contigs. Eleven of these were considered non-synonymous. The annotated transcriptome described in this study will be a useful basis for future work investigating the genetic response of abalone to stress.

  19. Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Koizumi, Shinya; Ohama, Naohiko; Mizoi, Junya [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shinozaki, Kazuo [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045 (Japan); Yamaguchi-Shinozaki, Kazuko, E-mail: akys@mail.ecc.u-tokyo.ac.jp [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-07-18

    Highlights: • HKL, a Hikeshi homologous gene is identified in Arabidopsis. • HKL interacts with two HSP70 isoforms and regulates the subcellular localization of HSC70-1. • The two HSP70 translocate into nucleus in response to heat stress. • Overexpression of HKL confers thermotolerance in transgenic plants. - Abstract: Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

  20. Adaptive capability as indicated by behavioral and physiological responses, plasma HSP70 level, and PBMC HSP70 mRNA expression in Osmanabadi goats subjected to combined (heat and nutritional) stressors

    Science.gov (United States)

    Shilja, Shaji; Sejian, V.; Bagath, M.; Mech, A.; David, C. G.; Kurien, E. K.; Varma, Girish; Bhatta, Raghavendra

    2016-09-01

    A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C ( n = 6; control), HS ( n = 6; heat stress), NS ( n = 6; nutritional stress), and CS ( n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly ( P < 0.01) between ad libitum fed groups (C and HS) and restricted feeding groups (NS and CS). The highest ( P < 0.01) lying time was recorded in the CS group while the lowest in the C and HS groups. The highest ( P < 0.01) drinking frequency was also recorded in the CS group. Water intake recorded was significantly ( P < 0.01) higher in both the HS and CS groups. The highest respiration rate (RR), pulse rate (PR), and rectal temperature (RT) during the afternoon were also recorded in the CS group. Further, skin temperature of the head, flank, and scrotum during the afternoon was also higher ( P < 0.01) in the CS group. In addition, both plasma HSP70 concentration and PBMC HSP70 messenger RNA (mRNA) transcript expression were also significantly ( P < 0.01) higher in the CS group. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the

  1. Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

    Science.gov (United States)

    MacLellan, Robyn J; Tunnah, Louise; Barnett, David; Wright, Patricia A; MacCormack, Tyson; Currie, Suzanne

    2015-10-01

    Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

  2. Energy sources and levels influenced on performance parameters, thyroid hormones, and HSP70 gene expression of broiler chickens under heat stress.

    Science.gov (United States)

    Raghebian, Majid; Sadeghi, Ali Asghar; Aminafshar, Mehdi

    2016-12-01

    The present study was conducted to evaluate the effects of energy sources and levels on body and organs weights, thyroid hormones, and heat shock protein (HSP70) gene expression in broilers under heat stress. In a completely randomized design, 600 1-day-old Cobb chickens were assigned to five dietary treatments and four replicates. The chickens were fed diet based on corn as main energy source and energy level based on Cobb standard considered as control (C), corn-based diet with 3 % lesser energy than the control (T1), corn-based diet with 6 % lesser energy than the control (T2), corn and soybean oil-based diet according to Cobb standard (T3), and corn and soybean oil-based diet with 3 % upper energy than the control (T4). Temperature was increased to 34 °C for 8 h daily from days 12 to 41 of age to induce heat stress. The chickens in T1 and T2 had lower thyroid hormones and corticosterone levels than those in C, T3, and T4. The highest liver weight was for C and the lowest one was for T4. The highest gene expression was found in chickens fed T4 diet, and the lowest gene expression was for those in T2 group. The highest feed intake and worse feed conversion ratio was related to chickens in T2. The chickens in T3 and T4 had higher feed intake and weight gain than those in C. The results showed that the higher energy level supplied from soybean oil could enhance gene expression of HSP70 and decline the level of corticosterone and thyroid hormones and consequently improved performance.

  3. Overexpression of GmHsp90s, a heat shock protein 90 (Hsp90 gene family cloning from soybean, decrease damage of abiotic stresses in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Jinyan Xu

    Full Text Available Hsp90 is one of the most conserved and abundant molecular chaperones and is an essential component of the protective stress response; however, its roles in abiotic stress responses in soybean (Glycine max remain obscure. Here, 12 GmHsp90 genes from soybean were identified and found to be expressed and to function differentially under abiotic stresses. The 12 GmHsp90 genes were isolated and named GmHsp90A1-GmHsp90A6, GmHsp90B1, GmHsp90B2, GmHsp90C1.1, GmHsp90C1.2, GmHsp90C2.1 and GmHsp90C2.2 based on their characteristics and high homology to other Hsp90s according to a new nomenclature system. Quantitative real-time PCR expression data revealed that all the genes exhibited higher transcript levels in leaves and could be strongly induced under heat, osmotic and salt stress but not cold stress. Overexpression of five typical genes (GmHsp90A2, GmHsp90A4, GmHsp90B1, GmHsp90C1.1 and GmHsp90C2.1 in Arabidopsis thaliana provided useful evidences that GmHsp90 genes can decrease damage of abiotic stresses. In addition, an abnormal accumulation of proline was detected in some transgenic Arabidopsis plants suggested overexpressing GmHsp90s may affect the synthesis and response system of proline. Our work represents a systematic determination of soybean genes encoding Hsp90s, and provides useful evidence that GmHsp90 genes function differently in response to abiotic stresses and may affect the synthesis and response system of proline.

  4. Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study.

    Science.gov (United States)

    Wang, Yongfei; Lin, Shoukai; Song, Qi; Li, Kuan; Tao, Huan; Huang, Jian; Chen, Xinhai; Que, Shufu; He, Huaqin

    2014-05-07

    Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors. In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities. Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.

  5. Modulation of ASK1 expression during overexpression of Trx and HSP70 in stressed fish liver mitochondria.

    Science.gov (United States)

    Padmini, Ekambaram; Vijaya Geetha, Bose

    2009-09-01

    Mitochondrial heat shock protein 70 (mtHSP70) is found to play a primary role in cellular defense against physiological stress like exposure to environmental contaminants and helpful in the maintenance of cellular homeostasis by promoting the cell survival. In the present investigation, the environmental-stress-induced increase in mtHSP70 levels along with the quantification of apoptosis signal regulating kinase 1 (ASK1) and thioredoxin (Trx) were measured in the liver mitochondria of grey mullets (Mugil cephalus) collected from the polluted Ennore estuary and the unpolluted Kovalam estuary for a period of 2 years. The results showed elevated lipid peroxide (LPO) and decreased total antioxidant capacity along with the decrease in mitochondrial viability percentage. Mitochondrial HSP70, ASK1, and Trx levels were increased under this stress condition. A 42% increase in LPO levels and 18% decrease in mitochondrial survivality were observed in the polluted-site fish liver mitochondria when compared to the results of unpolluted estuary. We also report that, under observed oxidative stress condition in Ennore fish samples, the ASK1 levels are only moderately elevated (13% increase). This may be due to mitochondrial-HSP70-induced adaptive tolerance signaling for the activation of Trx (22% increase) which suppresses the ASK1 expression thereby promoting the cell survival that leads to the maintenance of the cellular homeostasis.

  6. Reduced Hsp70 and Glutamine in Pediatric Severe Malaria Anemia

    DEFF Research Database (Denmark)

    Kempaiah, Prakasha; Dokladny, Karol; Karim, Zachary

    2016-01-01

    by decreased HSPA1A, a heat shock protein (Hsp) 70 coding gene. Hsp70 is a ubiquitous chaperone that regulates Nuclear Factor-kappa B (NF-κB) signaling and production of pro-inflammatory cytokines known to be important in malaria pathogenesis (e.g., IL-1β, IL-6 and TNF-α). Since the role of host Hsp70...... in malaria pathogenesis is unexplored, we investigated Hsp70 and molecular pathways in children with SMA. Validation experiments revealed that leukocytic HSP70 transcripts were reduced in SMA relative to non-severe malaria, and that intraleukocytic hemozoin (PfHz) was associated with lower HSP70. HSP70...... was correlated with reticulocyte production and Hb. Since glutamine (Gln) up-regulates Hsp70, modulates NF-κB activation, and attenuates over-expression of pro-inflammatory cytokines, circulating Gln was measured in children with malaria. Reduced Gln was associated with increased risk of developing SMA...

  7. HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

    Science.gov (United States)

    Cellura, Cinzia; Toubiana, Mylène; Parrinello, Nicolo; Roch, Philippe

    2006-01-01

    Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.

  8. Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

    Science.gov (United States)

    Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

    2011-01-01

    Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

  9. KADAR MDA DAN HSP 70 PADA PLASENTA PENDERITA PREEKLAMPSIA

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    Arleni

    2008-12-01

    Full Text Available The MDA and HSP70 Concentration in Preeclamptic Patient Placenta’s. Objective: Preeclampsia is a disease in pregnancy and characterized by hypertension and proteinuria. Preeclampsia and eclampsia are the most causes of maternal and fetal mortality and morbidity in Indonesia. Placental and systemic oxidative stress caused endothelial cell dysfunction and injury. Placental oxidative stress also linked to fetal growth restriction. HSP70 is essential for cellular recovery, survival and maintenance of homeostasis. The purpose of this study was to compare the MDA, a marker for oxidative stress and HSP70 production in placental of severe preeclampsia, mild preeclampsia and normotensive pregnant women. Placenta were collected after delivery from normotensive pregnancies (N=10, severe preeclampsia (N=10 and mild preeclampsia (N=10. Placenta was cultured in RPMI and 20% FBS, and supernatant were collected in day 3. MDA was measured using spectrophotometer and absorbance read in 530nm. HSP70 was measured using enzyme-linked immunosorbent assay. The mean MDA concentration did not differ significantly between patients with severe preeclampsia (7.13+5.36 nmol/ml and mild preeclampsia (4.82+2.47 nmol/ml when compared with normotensive pregnancies (4.57+2.4 nmol/ml. The mean HSP70 concentration in mild preeclampsia is highest (10.15+12.39 nmo/ml when compared with severe preeclampsia (3.78 +3.07 nmol/ml and normotensive pregnant women (3.76+4.65nmol/ml, but the difference was not significant. Although the difference was not significant, is indicates homeostasis response in mild preclampsia women is relative good. This response was abated in severe preeclampstic women. Although MDA and HSP70 concentration did not differ significantly between groups, however the high HSP70 concentration is indicates homeostasis response relatively good in mild preeclamptic women.

  10. Molecular characterization of three heat shock protein 70 genes and their expression profiles under thermal stress in the citrus red mite.

    Science.gov (United States)

    Yang, Li-Hong; Jiang, Hong-Bo; Liu, Yong-Hua; Dou, Wei; Wang, Jin-Jun

    2012-04-01

    Three heat shock protein 70 family transcripts, named PcHsp70-1, PcHsp70-2 and PcHsp70-3, were isolated from the citrus red mite, Panonychus citri. PcHsp70-1, PcHsp70-2, and PcHsp70-3 contained an open reading frame of 1977, 1968, and 2028 nucleotides that encoded 658, 655 and 675 amino acid residues, respectively. Comparison of deduced amino acid sequences of PcHsp70-1 and PcHsp70-2 showed 86.34% identity, while the amino acid sequence of PcHsp70-3 was only 57.39 and 58.75% identical to that of PcHsp70-1 and PcHsp70-2, respectively. Sequences and phylogenetic analyses suggested that PcHsp70-1 and PcHsp70-2 were cytosolic Hsps, whereas PcHsp70-3 was located in ER (endoplasmic reticulum). To accurately validate mRNA expression profiles of the three Hsp70s under thermal stress conditions, seven housekeeping genes were evaluated. Alpha-tubulin and RpII were selected as optimal endogenous references for cold shock and heat shock conditions, respectively. Real-time quantitative RT-PCR revealed that only the mRNA expression of PcHsp70-2 was up-regulated under heat shocks, and all of the three Hsp70s were constitutively expressed under cold shocks. The results suggest that the three Hsp70s were more critical to coping with heat than cold shocks.

  11. Genome-Wide Characterization of Heat-Shock Protein 70s from Chenopodium quinoa and Expression Analyses of Cqhsp70s in Response to Drought Stress.

    Science.gov (United States)

    Liu, Jianxia; Wang, Runmei; Liu, Wenying; Zhang, Hongli; Guo, Yaodong; Wen, Riyu

    2018-01-23

    Heat-shock proteins (HSPs) are ubiquitous proteins with important roles in response to biotic and abiotic stress. The 70-kDa heat-shock genes ( Hsp70s ) encode a group of conserved chaperone proteins that play central roles in cellular networks of molecular chaperones and folding catalysts across all the studied organisms including bacteria, plants and animals. Several Hsp70s involved in drought tolerance have been well characterized in various plants, whereas no research on Chenopodium quinoa HSPs has been completed. Here, we analyzed the genome of C. quinoa and identified sixteen Hsp70 members in quinoa genome. Phylogenetic analysis revealed the independent origination of those Hsp70 members, with eight paralogous pairs comprising the Hsp70 family in quinoa. While the gene structure and motif analysis showed high conservation of those paralogous pairs, the synteny analysis of those paralogous pairs provided evidence for expansion coming from the polyploidy event. With several subcellular localization signals detected in CqHSP70 protein paralogous pairs, some of the paralogous proteins lost the localization information, indicating the diversity of both subcellular localizations and potential functionalities of those HSP70s. Further gene expression analyses revealed by quantitative polymerase chain reaction (qPCR) analysis illustrated the significant variations of Cqhsp70s in response to drought stress. In conclusion, the sixteen Cqhsp70 s undergo lineage-specific expansions and might play important and varied roles in response to drought stress.

  12. Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner.

    Science.gov (United States)

    Amorós, M; Estruch, F

    2001-03-01

    Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes. Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection. Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation. Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.

  13. Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

    DEFF Research Database (Denmark)

    Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

    2010-01-01

    Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently...... reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism......-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds...

  14. Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

    Science.gov (United States)

    Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

    2016-05-01

    The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

  15. Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

    Directory of Open Access Journals (Sweden)

    Regina T Knapp

    Full Text Available Hsp70 binding protein 1 (HspBP1 and Bcl2-associated athanogene 1 (BAG-1, the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70 chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR, the mineralocorticoid receptor (MR, and the androgen receptor (AR. BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

  16. Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Gehrmann MK

    2015-09-01

    Full Text Available Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

  17. Genome-wide analysis of the potato Hsp20 gene family: identification, genomic organization and expression profiles in response to heat stress.

    Science.gov (United States)

    Zhao, Peng; Wang, Dongdong; Wang, Ruoqiu; Kong, Nana; Zhang, Chao; Yang, Chenghui; Wu, Wentao; Ma, Haoli; Chen, Qin

    2018-01-18

    Heat shock proteins (Hsps) are essential components in plant tolerance mechanism under various abiotic stresses. Hsp20 is the major family of heat shock proteins, but little of Hsp20 family is known in potato (Solanum tuberosum), which is an important vegetable crop that is thermosensitive. To reveal the mechanisms of potato Hsp20s coping with abiotic stresses, analyses of the potato Hsp20 gene family were conducted using bioinformatics-based methods. In total, 48 putative potato Hsp20 genes (StHsp20s) were identified and named according to their chromosomal locations. A sequence analysis revealed that most StHsp20 genes (89.6%) possessed no, or only one, intron. A phylogenetic analysis indicated that all of the StHsp20 genes, except 10, were grouped into 12 subfamilies. The 48 StHsp20 genes were randomly distributed on 12 chromosomes. Nineteen tandem duplicated StHsp20s and one pair of segmental duplicated genes (StHsp20-15 and StHsp20-48) were identified. A cis-element analysis inferred that StHsp20s, except for StHsp20-41, possessed at least one stress response cis-element. A heatmap of the StHsp20 gene family showed that the genes, except for StHsp20-2 and StHsp20-45, were expressed in various tissues and organs. Real-time quantitative PCR was used to detect the expression level of StHsp20 genes and demonstrated that the genes responded to multiple abiotic stresses, such as heat, salt or drought stress. The relative expression levels of 14 StHsp20 genes (StHsp20-4, 6, 7, 9, 20, 21, 33, 34, 35, 37, 41, 43, 44 and 46) were significantly up-regulated (more than 100-fold) under heat stress. These results provide valuable information for clarifying the evolutionary relationship of the StHsp20 family and in aiding functional characterization of StHsp20 genes in further research.

  18. Characterization of heat shock protein 70 transcript from Nilaparvata lugens (Stål): Its response to temperature and insecticide stresses.

    Science.gov (United States)

    Lu, Kai; Chen, Xia; Liu, Wenting; Zhang, Zhichao; Wang, Ying; You, Keke; Li, Yue; Zhang, Rongbin; Zhou, Qiang

    2017-10-01

    The brown planthopper, Nilaparvata lugens, possesses a strong adaptability to extreme temperature and insecticide stresses. Heat shock proteins (Hsps) are highly conserved molecular chaperones and play a pivotal role in response to various environmental stresses in insects. However, little is known about the response of Hsps to stresses in N. lugens. In the present study, an inducible Hsp70 (NlHsp70) was isolated from this insect and transcriptional expression patterns of NlHsp70 under temperature and insecticide stresses were analyzed. The full-length of NlHsp70 was 2805bp with an open reading frame (ORF) of 1896bp, showing high homology to its counterparts in other species. Expression of NlHsp70 was not altered by heat shock for 1h, nor following recovery from thermal stress. Conversely, decreased expression of NlHsp70 was observed in response to cold shock. In addition, the expression of NlHsp70 increased after imidacloprid exposure. RNA interference experiment combined with insecticide injury assay also demonstrated that NlHsp70 was essential for resistance against insecticide exposure. These observations indicated that NlHsp70 was an important gene involved in the resistance or tolerance to environmental stresses in N. lugens. Interestingly, weak changes in mRNA expression levels of two thermal-inducible Hsp genes, NlHsp90 and NlHsc70 were observed in imidacloprid-exposed N. lugens adults, suggesting that different Hsps may respond differential to the extreme temperature and insecticide stresses. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Genetics of human longevity with emphasis on the relevance of HSP70 as candidate genes

    DEFF Research Database (Denmark)

    Singh, Ripudaman; Kølvrå, Steen; Rattan, Suresh I S

    2007-01-01

    Human longevity is determined to a certain extent by genetic factors. Several candidate genes have been studied for their association with human longevity, but the data collected so far are inconclusive. One of the reasons is the choice of the candidate genes in addition to the choice...... of an appropriate study design and methodology. Since aging is characterized by a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, the focus of studies on human longevity association with genes has now shifted to the pathways of cellular maintenance and repair...... mechanisms. One such pathway includes the battery of stress response genes, especially the heat shock protein HSP70 genes. Three such genes, HSPA1A, HSPA1B and HSPA1L, are present within the MHC-III region on the short arm of chromosome 6. We and others have found alleles, genotypes and haplotypes which have...

  20. Fine particulate matter potentiates type 2 diabetes development in high-fat diet-treated mice: stress response and extracellular to intracellular HSP70 ratio analysis.

    Science.gov (United States)

    Goettems-Fiorin, Pauline Brendler; Grochanke, Bethânia Salamoni; Baldissera, Fernanda Giesel; Dos Santos, Analu Bender; Homem de Bittencourt, Paulo Ivo; Ludwig, Mirna Stela; Rhoden, Claudia Ramos; Heck, Thiago Gomes

    2016-12-01

    Exposure to fine particulate matter (PM 2.5 ) air pollution is a risk factor for type 2 diabetes (T2DM). We argue whether the potentiating effect of PM 2.5 over the development of T2DM in high-fat diet (HFD)-fed mice would be related to modification in cell stress response, particularly in antioxidant defenses and 70-kDa heat shock proteins (HSP70) status. Male mice were fed standard chow or HFD for 12 weeks and then randomly exposed to daily nasotropic instillation of PM 2.5 for additional 12 weeks under the same diet schedule, divided into four groups (n = 14-15 each): Control, PM 2.5 , HFD, and HFD + PM 2.5 were evaluated biometric and metabolic profiles of mice, and cellular stress response (antioxidant defense and HSP70 status) of metabolic tissues. Extracellular to intracellular HSP70 ratio ([eHSP72]/[iHSP70]), viz. H-index, was then calculated. HFD + PM 2.5 mice presented a positive correlation between adiposity, increased body weight and glucose intolerance, and increased glucose and triacylglycerol plasma levels. Pancreas exhibited lower iHSP70 expression, accompanied by 3.7-fold increase in the plasma to pancreas [eHSP72]/[iHSP70] ratio. Exposure to PM 2.5 markedly potentiated metabolic dysfunction in HFD-treated mice and promoted relevant alteration in cell stress response assessed by [eHSP72]/[iHSP70], a relevant biomarker of chronic low-grade inflammatory state and T2DM risk.

  1. Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene

    International Nuclear Information System (INIS)

    Li, G.C.; Li, Ligeng; Liu, Yunkang; Mak, J.Y.; Chen, Lili; Lee, W.M.F.

    1991-01-01

    The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. The authors have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, low-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45C and their thermal survivals were determined by colony-formation assay, they found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress

  2. Non-invasive assessment of animal exercise stress: real-time PCR of GLUT4, COX2, SOD1 and HSP70 in avalanche military dog saliva.

    Science.gov (United States)

    Diverio, S; Guelfi, G; Barbato, O; Di Mari, W; Egidi, M G; Santoro, M M

    2015-01-01

    Exercise has been shown to increase mRNA expression of a growing number of genes. The aim of this study was to assess if mRNA expression of the metabolism- and oxidative stress-related genes GLUT4 (glucose transporter 4), COX2 (cyclooxygenase 2), SOD1 (superoxide dismutase 1) and HSP70 (heat shock protein 70) in saliva changes following acute exercise stress in dogs. For this purpose, 12 avalanche dogs of the Italian Military Force Guardia di Finanza were monitored during simulation of a search for a buried person in an artificial avalanche area. Rectal temperature (RT) and saliva samples were collected the day before the trial (T0), immediately after the descent from a helicopter at the onset of a simulated avalanche search and rescue operation (T1), after the discovery of the buried person (T2) and 2 h later (T3). Expressions of GLUT4, SOD1, COX2 and HSP70 were measured by real-time PCR. The simulated avalanche search and rescue operation was shown to exert a significant effect on RT, as well as on the expression of all metabolism- and oxidative stress-related genes investigated, which peaked at T2. The observed expression patterns indicate an acute exercise stress-induced upregulation, as confirmed by the reductions in expression at T3. Moreover, our findings indicate that saliva is useful for assessing metabolism- and oxidative stress-related genes without the need for restraint, which could affect working dog performance.

  3. Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding

    International Nuclear Information System (INIS)

    Guinez, Celine; Mir, Anne-Marie; Leroy, Yves; Cacan, Rene; Michalski, Jean-Claude; Lefebvre, Tony

    2007-01-01

    Numerous recent works strengthen the idea that the nuclear and cytosolic-specific O-GlcNAc glycosylation protects cells against injuries. We have first investigated O-GlcNAc level and Hsp70-GlcNAc-binding activity (HGBA) behaviour after exposure of HeLa and HepG 2 cells to a wide variety of stresses. O-GlcNAc and HGBA responses were different according to the stress and according to the cell. HGBA was released for almost all stresses, while O-GlcNAc level was modified either upwards or downwards, depending to the stress. Against all expectations, we demonstrated that energy charge did not significantly vary with stress whereas UDP-GlcNAc pools were more dramatically affected even if differences in UDP-GlcNAc contents were not correlated with O-GlcNAc variations suggesting that O-GlcNAc transferase is itself finely regulated during cell injury. Finally, HGBA could be triggered by proteasome inhibition and by L-azetidine-2-carboxylic acid (a proline analogue) incorporation demonstrating that protein misfolding is one of the key-activator of this Hsp70 property

  4. Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

    Science.gov (United States)

    Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

    2005-06-01

    To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

  5. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    Heat shock proteins (HSPs) are highly conserved molecules, which support folding of proteins under physiological conditions and mediate protection against lethal damage after various stress stimuli. Five HSP families exist defined by their molecular size (i.e. HSP100, HSP90, HSP70, HSP60, and the......Heat shock proteins (HSPs) are highly conserved molecules, which support folding of proteins under physiological conditions and mediate protection against lethal damage after various stress stimuli. Five HSP families exist defined by their molecular size (i.e. HSP100, HSP90, HSP70, HSP60...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...... hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 surface expression was confined to the apoptotic Annexin V positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis...

  6. Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica.

    Science.gov (United States)

    Martynova, M G; Bystrova, O A; Shabelnikov, S V; Margulis, B A; Prokofjeva, D S

    2007-04-01

    Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.

  7. Expression profile of HSP genes during different seasons in goats (Capra hircus).

    Science.gov (United States)

    Dangi, Satyaveer Singh; Gupta, Mahesh; Maurya, Divakar; Yadav, Vijay Prakash; Panda, Rudra Prasanna; Singh, Gyanendra; Mohan, Nitai Haridas; Bhure, Sanjeev Kumar; Das, Bikash Chandra; Bag, Sadhan; Mahapatra, Ramkrishna; Taru Sharma, Guttalu; Sarkar, Mihir

    2012-12-01

    The present study has demonstrated the expression of HSP60, HSP70, HSP90, and UBQ in peripheral blood mononuclear cells (PBMCs) during different seasons in three different age groups (Groups I, II, and III with age of 0-2, 2-5, and >5 years, respectively) of goats of tropical and temperate regions. Real-time polymerase chain reaction was applied to investigate mRNA expression of examined factors. Specificity of the desired products was documented using analysis of the melting temperature and high-resolution gel electrophoresis to verify that the transcripts are of the exact molecular size predicted. The mRNA expression of HSP60, HSP90, and UBQ was significantly higher (P tropical and temperate region goats. HSP70 mRNA expression was significantly higher (P tropical region goats. However, in the temperate region, in goats from all the three age groups studied, a non-significant difference of HSP70 expression between summer and winter seasons was noticed. In conclusion, results demonstrate that (1) HSP genes are expressed in caprine PBMCs and (2) higher expression of HSPs during thermal stress suggest possible involvement of them to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis in goats.

  8. Genome-wide analysis of the CaHsp20 gene family in pepper: comprehensive sequence and expression profile analysis under heat stress

    Directory of Open Access Journals (Sweden)

    Meng eGuo

    2015-10-01

    Full Text Available The Hsp20 genes are present in all plant species and play important roles in alleviating heat stress and enhancing plant thermotolerance by preventing the irreversible aggregation of denaturing proteins. However, very little is known about the CaHsp20 gene family in pepper (Capsicum annuum L., an important vegetable crop with character of temperate but thermosensitive. In this study, a total of 35 putative pepper Hsp20 genes (CaHsp20s were identified and renamed on the basis of their molecular weight, and then their gene structure, genome location, gene duplication, phylogenetic relationship and interaction network were also analyzed. The expression patterns of CaHsp20 genes in four different tissues (root, stem, leaf and flower from the thermotolerant line R9 under heat stress condition were measured using semi-quantitative RT-PCR. The transcripts of most CaHsp20 genes maintained a low level in all of the four tissues under normal temperature condition, but were highly induced by heat stress, while the expression of CaHsp16.6b, 16.7 and 23.8 were only detected in specific tissues and were not so sensitive to heat stress like other CaHsp20 genes. In addition, compared to those in thermotolerant line R9, the expression peak of most CaHsp20 genes in thermosensitive line B6 under heat stress was hysteretic, and several CaHsp20 genes (CaHsp16.4, 18.2a, 18.7, 21.2, 22.0, 25.8 and 25.9 showed higher expression levels in both line B6 and R9. These data suggest that the CaHsp20 genes may be involved in heat stress and defense responses in pepper, which provides the basis for further functional analyses of CaHsp20s in the formation of pepper acquired thermotoleance.

  9. Response of heat shock protein genes of the oriental fruit moth under diapause and thermal stress reveals multiple patterns dependent on the nature of stress exposure.

    Science.gov (United States)

    Zhang, Bo; Peng, Yu; Zheng, Jincheng; Liang, Lina; Hoffmann, Ary A; Ma, Chun-Sen

    2016-07-01

    Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.

  10. Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

    Science.gov (United States)

    Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

    2017-08-01

    Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

  11. Molecular cloning of a Candida albicans gene (SSB1) coding for a protein related to the Hsp70 family.

    Science.gov (United States)

    Maneu, V; Cervera, A M; Martinez, J P; Gozalbo, D

    1997-06-15

    We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2.1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans.

  12. Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study.

    Directory of Open Access Journals (Sweden)

    Saurabh Gupta

    Full Text Available Heat shock protein 70 (HSP70 is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70 has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state and with HSP70 protein of E. coli 70kDa DnaK (close state and relaxed them for 100 nanoseconds (ns using all-atom Molecular Dynamics (MD Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA and Minimum Distance Matrix (MDM. The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.

  13. Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

    Directory of Open Access Journals (Sweden)

    VIVIAN WILHELM

    2005-01-01

    Full Text Available We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60 and 70 (Hsp70 of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.

  14. Transcriptome, expression, and activity analyses reveal a vital heat shock protein 70 in the stress response of stony coral Pocillopora damicornis.

    Science.gov (United States)

    Zhang, Yidan; Zhou, Zhi; Wang, Lingui; Huang, Bo

    2018-02-12

    Coral bleaching occurs worldwide with increasing frequencies and intensities, which is caused by the stress response of stony coral to environmental change, especially increased sea surface temperature. In the present study, transcriptome, expression, and activity analyses were employed to illustrate the underlying molecular mechanisms of heat shock protein 70 (HSP70) in the stress response of coral to environmental changes. The domain analyses of assembled transcripts revealed 30 HSP70 gene contigs in stony coral Pocillopora damicornis. One crucial HSP70 (PdHSP70) was observed, whose expressions were induced by both elevated temperature and ammonium after expression difference analysis. The complete complementary DNA (cDNA) sequence of PdHSP70 was identified, which encoded a polypeptide of 650 amino acids with a molecular weight of 71.93 kDa. The deduced amino acid sequence of PdHSP70 contained a HSP70 domain (from Pro8 to Gly616), and it shared the highest similarity (95%) with HSP70 from Stylophora pistillata. The expression level of PdHSP70 gene increased significantly at 12 h, and returned to the initial level at 24 h after the stress of high temperature (32 °C). The cDNA fragment encoding the mature peptide of PdHSP70 was recombined and expressed in the prokaryotic expression system. The ATPase activity of recombinant PdHSP70 protein was determined, and it did not change significantly in a wide range of temperature from 25 to 40 °C. These results collectively suggested that PdHSP70 was a vital heat shock protein 70 in the stony coral P. damicornis, whose mRNA expression could be induced by diverse environmental stress and whose activity could remain stable under heat stress. PdHSP70 might be involved in the regulation of the bleaching owing to heat stress in the stony coral P. damicornis.

  15. Epigallocatechin-3-gallate suppresses the expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo

    International Nuclear Information System (INIS)

    Tran, Phan LCHB; Kim, Soo-A; Choi, Hong Seok; Yoon, Jung-Hoon; Ahn, Sang-Gun

    2010-01-01

    Epigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression. Cell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model. Treatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H 2 O 2 , 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression. Overall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer

  16. The role of HSP70 in mediating age-dependent mortality in sepsis

    Science.gov (United States)

    McConnell, Kevin W.; Fox, Amy C.; Clark, Andrew T.; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A.; Farris, Alton B.; Buchman, Timothy G.; Hunt, Clayton R.; Coopersmith, Craig M.

    2011-01-01

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6–12week old) and aged (16–17 month old) HSP70−/− and wild type (WT) mice to determine if HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture (CLP), Pseudomonas aeruginosa pneumonia or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to CLP (p=0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared to WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/−mice had increased systemic levels of TNF-α, IL-6, IL-10 and IL-1β compared to WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation. PMID:21296977

  17. Association of HSP70 and its co-chaperones with Alzheimer's disease

    NARCIS (Netherlands)

    L. Broer (Linda); M.A. Ikram (Arfan); M. Schuur (Maaike); A.L. DeStefano (Anita); J.C. Bis (Joshua); F. Liu (Fan); F. Rivadeneira Ramirez (Fernando); A.G. Uitterlinden (André); A. Beiser (Alexa); W.T. Longstreth Jr; A. Hofman (Albert); Y.S. Aulchenko (Yurii); S. Seshadri (Sudha); A.L. Fitzpatrick (Annette); B.A. Oostra (Ben); M.M.B. Breteler (Monique); P. Tikka-Kleemola (Päivi)

    2011-01-01

    textabstractThe heat shock protein (HSP) 70 family has been implicated in the pathology of Alzheimer's disease (AD). In this study, we examined common genetic variations in the 80 genes encoding HSP70 and its co-chaperones. We conducted a study in a series of 462 patients and 5238 unaffected

  18. The stress-induced heat shock protein 70.3 expression is regulated by a dual-component mechanism involving alternative polyadenylation and HuR.

    Science.gov (United States)

    Kraynik, Stephen M; Gabanic, Andrew; Anthony, Sarah R; Kelley, Melissa; Paulding, Waltke R; Roessler, Anne; McGuinness, Michael; Tranter, Michael

    2015-06-01

    Heat shock protein 70.3 (Hsp70.3) expression increases in response to cellular stress and plays a cytoprotective role. We have previously shown that Hsp70.3 expression is controlled through coordinated post-transcriptional regulation by miRNAs and alternative polyadenylation (APA), and APA-mediated shortening of the Hsp70.3 3'-UTR facilitates increased protein expression. A stress-induced increase in Hsp70.3 mRNA and protein expression is accompanied by alternative polyadenylation (APA)-mediated truncation of the 3'UTR of the Hsp70.3 mRNA transcript. However, the role that APA plays in stress-induced expression of Hsp70.3 remains unclear. Our results show that APA-mediated truncation of the Hsp70.3 3'UTR increases protein expression through enhanced polyribosome loading. Additionally, we demonstrate that the RNA binding protein HuR, which has been previously shown to play a role in mediating APA, is necessary for heat shock mediated increase in Hsp70.3 mRNA and protein. However, it is somewhat surprising to note that HuR does not play a role in APA of the Hsp70.3 mRNA, and these two regulatory events appear to be mutually exclusive regulators of Hsp70.3 expression. These results not only provide important insight to the regulation of stress response genes following heat shock, but also contribute an enhanced understanding of how alternative polyadenylation contributes to gene regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  20. Examination the expression pattern of HSP70 heat shock protein in chicken PGCs and developing genital ridge

    Directory of Open Access Journals (Sweden)

    Mahek Anand

    2016-05-01

    Full Text Available Chicken Primordial Germ cells (PGCs are emerging pioneers in the field of applied embryology and stem cell technology. Now-a-days transgenic chickens are promising models to study human disease pathophysiology and drug designing. However, most of the molecular mechanism, which govern the stemness and pluripotency of chicken PGCs, not known in details. Recent studies have indicated the role of HSP70 in early embryonic development in many vertebrate species. Exposure of chicken to heat stress result in activation of heat shock factors which activate the transcription of HSP70. Exposure chicken eggs to acute heat stress effects HSP70 expression in PGCs and gonads. HSP70 helps in maintaining the integrity of chicken PGCs. A new emerging role of HSP70 in apoptosis has emerged. In our lab, we aim to characterize the expression of cHsp70 in chicken PGCs and gonads during embryonic development by subjecting the parents to acute levels of heat stress. Chickens whose parents subjected to heat stress showed varied expression of cHsp70 and also improved thermo tolerance. In the future we plan to study other factors and miRNAs, which is characterized as an emerging player in regulating heat shock protein response in chicken and also plays an important role in apoptosis.

  1. Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

    Science.gov (United States)

    Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

    Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

  2. HDJC9, a novel human type C DnaJ/HSP40 member interacts with and cochaperones HSP70 through the J domain

    International Nuclear Information System (INIS)

    Han Chaofeng; Chen Taoyong; Li Nan; Yang Mingjin; Wan Tao; Cao Xuetao

    2007-01-01

    HSP40s are a subfamily of heat shock proteins (HSPs) and play important roles in regulation of cell proliferation, survival and apoptosis by serving as chaperones for HSP70s. Up to date hundreds of HSP40 proteins derived from various species ranging from Escherichia coli to homo sapiens have been identified. Here we report the cloning and characterization of a novel human type C DnaJ homologue, HDJC9, containing a typical N-terminal J domain. HDJC9 is upregulated at both mRNA and protein levels upon various stress and mitogenic stimulations. HDJC9 is mainly localized in cell nuclei under normal culture conditions while it is transported into cytoplasm and plasma membrane upon heat shock stress through a non-classical and lipid-dependent pathway. HDJC9 can interact with HSP70s and activate the ATPase activity of HSP70s, both of which are dependent on the J domain. Our data suggest that HDJC9 is a novel cochaperone for HSP70s

  3. Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat.

    Science.gov (United States)

    Muthusamy, Senthilkumar K; Dalal, Monika; Chinnusamy, Viswanathan; Bansal, Kailash C

    2017-04-01

    Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under

  4. Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

    Directory of Open Access Journals (Sweden)

    Hicham Mahboubi

    2015-12-01

    Full Text Available Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.Methods and Results. Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i the physiological state of the cell and (ii the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.Discussion. Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.

  5. Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera inhabiting thermally contrasting environments

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    Bettencourt Brian R

    2011-03-01

    Full Text Available Abstract Background Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Results Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. Conclusions The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form

  6. ANTI-HSP60 and ANTI-HSP70 antibody levels and micro/ macrovascular complications in type 1 diabetes: the EURODIAB Study

    DEFF Research Database (Denmark)

    Gruden, G.; Bruno, G.; Chaturvedi, N.

    2009-01-01

    OBJECTIVES: The heat shock proteins 60 and 70 (HSP60, HSP70) play an important role in cytoprotection. Under stress conditions they are released into the circulation and elicit an immune response. Anti-HSP60 and anti-HSP70 antibody levels have been associated with cardiovascular disease. Type 1......-control study from the EURODIAB Study of 531 type 1 diabetic patients was performed. SUBJECTS: Cases (n = 363) were defined as those with one or more complications of diabetes; control subjects (n = 168) were all those with no evidence of any complication. We measured anti-HSP60 and anti-HSP70 antibody levels...... quartiles were associated with a 47% reduced odds ratio of micro/macrovascular complications, independently of conventional risk factors, markers of inflammation and endothelial dysfunction [odds ratio (OR) = 0.53, 95% confidence intervals (CI): 0.28-1.02]. CONCLUSIONS: In this large cohort of type 1...

  7. HSP70 expression in the copper butterfly Lycaena tityrus across altitudes and temperatures

    DEFF Research Database (Denmark)

    Karl, I.; Sørensen, Jesper Givskov; Loeschcke, Volker

    2009-01-01

    temperatures show differences in HSP70 expression. HSP70 expression increased substantially at the higher rearing temperature in low-altitude butterflies, which might represent an adaptation to occasionally occurring heat spells. On the other hand, high-altitude butterflies showed much less plasticity...... in response to rearing temperatures, and overall seem to rely more on genetically fixed thermal stress resistance. Whether the latter indicates a higher vulnerability of high-altitude populations to global warming needs further investigation. HSP70 expression increased with both colder and warmer induction......The ability to express heat-shock proteins (HSP) under thermal stress is an essential mechanism for ectotherms to cope with unfavourable conditions. In this study, we investigate if Copper butterflies originating from different altitudes and/or being exposed to different rearing and induction...

  8. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

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    Magdalena Wisniewska

    2010-01-01

    Full Text Available The 70-kDa heat shock proteins (Hsp70 are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD with ATPase activity, and a C-terminal substrate binding domain (SBD. We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  9. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

    Science.gov (United States)

    Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

    2010-01-11

    The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  10. First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

    Science.gov (United States)

    Colson-Proch, Céline; Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

    2010-05-01

    Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

  11. Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

    Science.gov (United States)

    Shorbagi, Sadek; Brown, Ian R

    2016-11-01

    Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

  12. HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

    Science.gov (United States)

    Tsuchida, Shinji; Arai, Yuji; Takahashi, Kenji A; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Inoue, Hiroaki; Ikoma, Kazuya; Ueshima, Keiichiro; Matsuki, Tomohiro; Mazda, Osam; Kubo, Toshikazu

    2014-08-01

    We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1α)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2 , which induces HIF-1α, to simulate hypoxia, or transfected with siRNAs targeting HIF-1α (si-HIF-1α) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1α under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1α. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1α or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1α or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1α or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1α-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF-1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes

    Science.gov (United States)

    Kimura, Richard H.; Choudary, Prabhakara V.; Stone, Koni K.; Schmid, Carl W.

    2001-01-01

    This study surveys the induction of RNA polymerase III (Pol III)–directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III–directed transcription. PMID:11599568

  14. Progranulin Recruits HSP70 to ?-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease

    OpenAIRE

    Jian, Jinlong; Tian, Qing-Yun; Hettinghouse, Aubryanna; Zhao, Shuai; Liu, Helen; Wei, Jianlu; Grunig, Gabriele; Zhang, Wujuan; Setchell, Kenneth D.R.; Sun, Ying; Overkleeft, Herman S.; Chan, Gerald L.; Liu, Chuan-ju

    2016-01-01

    Highlights ? PGRN is required for lysosomal appearance of GCase and PGRN deficiency causes GCase/LIMP2 aggregation upon stress ? PGRN directly binds to GCase through a two-site mechanism ? PGRN recruits HSP70 to GCase and prevents GCase aggregation in response to stress ? PGRN derivative Pcgin binds to GCase and HSP70 and is therapeutic against Gaucher disease In this study, we demonstrate that PGRN directly binds to GCase and is required for the lysosomal appearance of GCase. In addition, HS...

  15. A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

    Science.gov (United States)

    Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

    2016-01-01

    Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

  16. Structure of a new crystal form of human Hsp70 ATPase domain.

    Science.gov (United States)

    Osipiuk, J; Walsh, M A; Freeman, B C; Morimoto, R I; Joachimiak, A

    1999-05-01

    Hsp70 proteins are highly conserved proteins induced by heat shock and other stress conditions. An ATP-binding domain of human Hsp70 protein has been crystallized in two major morphological forms at pH 7.0 in the presence of PEG 8000 and CaCl2. Both crystal forms belong to the orthorhombic space group P212121, but show no resemblance in unit-cell parameters. Analysis of the crystal structures for both forms shows a 1-2 A shift of one of the subdomains of the protein. This conformational change could reflect a 'natural' flexibility of the protein which might be relevant to ATP binding and may facilitate the interaction of other proteins with Hsp70 protein.

  17. Imbalance of Hsp70 family variants fosters tau accumulation.

    Science.gov (United States)

    Jinwal, Umesh K; Akoury, Elias; Abisambra, Jose F; O'Leary, John C; Thompson, Andrea D; Blair, Laura J; Jin, Ying; Bacon, Justin; Nordhues, Bryce A; Cockman, Matthew; Zhang, Juan; Li, Pengfei; Zhang, Bo; Borysov, Sergiy; Uversky, Vladimir N; Biernat, Jacek; Mandelkow, Eckhard; Gestwicki, Jason E; Zweckstetter, Markus; Dickey, Chad A

    2013-04-01

    Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants

  18. The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Fanxing; Tee, Catherine; Liu, Michelle [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Sherry, James P. [Aquatic Contaminants Research Division, Environment Canada, Burlington, Ontario L7R 4A6 (Canada); Dixon, Brian; Duncker, Bernard P. [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Bols, Niels C., E-mail: ncbols@uwaterloo.ca [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada)

    2014-01-15

    Highlights: •2-Phenylethynesulfonamide (PES) is an inhibitor of p53 and HSP 70 in mammals. •In the fish epithelial cell line, RTgill-W1, PES enhanced ROS generation and was cytotoxic. •RTgill-W1 death was by apoptosis and blocked by the anti-oxidant N-acetylcysteine. •This is the first report linking PES-induced cell death to ROS. •With this background PES should be useful for studying fish cell survival pathways. -- Abstract: The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24 h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24 h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.

  19. Genome-wide identification and expression profiling of tomato Hsp20 gene family in response to biotic and abiotic stresses

    Directory of Open Access Journals (Sweden)

    jiahong yu

    2016-08-01

    Full Text Available The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps in plants, but little is known about this family in tomato (Solanum lycopersicum, an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20 gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83% were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root and reproductive organs (floral bud and flower, suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript

  20. Genome-Wide Identification and Expression Profiling of Tomato Hsp20 Gene Family in Response to Biotic and Abiotic Stresses.

    Science.gov (United States)

    Yu, Jiahong; Cheng, Yuan; Feng, Kun; Ruan, Meiying; Ye, Qingjing; Wang, Rongqing; Li, Zhimiao; Zhou, Guozhi; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

    2016-01-01

    The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps) in plants, but little is known about this family in tomato (Solanum lycopersicum), an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20) gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship, and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83%) were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root) and reproductive organs (floral bud and flower), suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript levels of SlHsp20

  1. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

    International Nuclear Information System (INIS)

    Gilmour, D.S.; Lis, J.T.

    1986-01-01

    By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

  2. Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

    Science.gov (United States)

    Weiss, Yoram G; Maloyan, Alina; Tazelaar, John; Raj, Nichelle; Deutschman, Clifford S

    2002-09-01

    The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

  3. Expression of HSP70 in cerebral ischemia and neuroprotetive action of hypothermia and ketoprofen Expressão de HSP70 na isquemia cerebral e a ação neuroprotetora da hipotermia e do cetoprofeno

    Directory of Open Access Journals (Sweden)

    Daniela Pretti da Cunha Tirapelli

    2010-08-01

    Full Text Available Heat shock proteins (HSPs are molecular chaperones that bind to other proteins to shepherd them across membranes and direct them to specific locations within a cell. Several injurious stimuli can induce Hsp70 expression, including ischemia. This study aimed to investigate the pattern of expression of protein (immunohistochemistry and gene (real-time PCR Hsp70 in experimental focal cerebral ischemia in rats by occlusion of the middle cerebral artery for 1 hour and the role of neuroprotection with hypothermia (H and ketoprofen (K. The infarct volume was measured using morphometric analysis defined by triphenyl tetrazolium chloride. It was observed increases in the protein (p=0.0001 and gene (p=0.0001 Hsp70 receptor in the ischemic areas that were reduced by H (protein and gene: pProteínas de choque térmico (HSPs são chaperones moleculares que se ligam a outras proteínas para atravessar as membranas e encaminhá-las para locais específicos dentro de uma célula. Vários estímulos nocivos podem induzir a expressão de Hsp70, incluindo isquemia. Este estudo teve como objetivo investigar o padrão de expressão protéica (imunohistoquímica e gênica (PCR em tempo real de Hsp70 na isquemia cerebral focal experimental em ratos pela oclusão da artéria cerebral média durante 1 hora e o papel da neuroproteção com hipotermia (H e cetoprofeno (C. O volume de infarto foi calculado através da análise morfométrica definido por cloreto de trifenil tetrazólio. Foi observado aumento na expressão proteína (p=0,0001 e gênica (p=0,0001 de Hsp70 nas áreas isquêmicas que foram reduzidas pela H (proteína e gene: p<0,05, C (proteína: p<0,001 e H+K (proteína: p<0,01 e gene: p<0,05. O aumento de Hsp70 na área isquêmica sugere que a neuroexcitotoxicidade mediada pela Hsp70 desempenha um papel importante na morte celular e que o efeito neuroprotetor tanto da H quanto do C está diretamente envolvido com a Hsp70.

  4. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    Directory of Open Access Journals (Sweden)

    Yuping Zhang

    Full Text Available Heat shock proteins (Hsps are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78, Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h, were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

  5. Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

    Science.gov (United States)

    Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2015-01-01

    Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

  6. Transcription of four Rhopalosiphum padi (L.) heat shock protein genes and their responses to heat stress and insecticide exposure.

    Science.gov (United States)

    Li, Yuting; Zhao, Qi; Duan, Xinle; Song, Chunman; Chen, Maohua

    2017-03-01

    The bird cherry-oat aphid, Rhopalosiphum padi (L.), a worldwide destructive pest, is more heat tolerant than other wheat aphids, and it has developed resistance to different insecticides. Heat shock proteins (HSPs) play an important role in coping with environmental stresses. To investigate Hsp transcriptional responses to heat and insecticide stress, four full-length Hsp genes from R. padi (RpHsp60, RpHsc70, RpHsp70-1, and RpHsp70-2) were cloned. Four RpHsps were expressed during all R. padi developmental stages, but at varying levels. The mRNA levels of RpHsps were increased under thermal stress and reached maximal induction at a lower temperature (36°C) in the alate morph than in the apterous morph (37°C or 38°C). RpHsp expressions under heat stress suggest that RpHsp70-1 and RpHsp70-2 are inducible in both apterous and alate morphs, RpHsc70 is only heat-inducible in apterous morph, and RpHsp60 exhibits poor sensitivity to heat stress. The pretreatment at 37°C significantly increase both the survival rate and the RpHsps expression level of R. padi at subsequent lethal temperature. Under exposure to two sublethal concentrations (LC 10 and LC 30 ) of beta-cypermethrin, both RpHsp70-1 and RpHsp70-2 expressions were induced and reached a maximum 24h after exposure. In contrast, expression of RpHsp60 was not induced by either sublethal concentration of beta-cypermethrin. Moreover, the responses of RpHsp70-1 and RpHsp70-2 to heat shock were more sensitive than those to beta-cypermethrin. These results suggest that induction of RpHsp expression is related to thermal tolerance, and that RpHsp70-1 and RpHsp70-2 are the primary genes involved in the response to both heat and pesticide stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

    Science.gov (United States)

    Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Yusuff, Oladosu A; Ismail, Mohd R; Miah, Gous

    2018-03-01

    Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F 1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC 1 F 1 , BC 2 F 1 and BC 3 F 1 . The average recipient allele of the selected four BC 1 F 1 plants was 80.75% which were used to produce the BC 2 F 1 generation. BC 1 -P 7 was the best BC 1 F 1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC 3 F 1 was 95.37%. Hsp gene expression analysis was carried out on BC 1 F 1 , BC 2 F 1 and BC 3 F 1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent

  8. Cytosolic Hsp70/Hsc70 protein expression in lymphocytes exposed to low gamma-ray dose

    International Nuclear Information System (INIS)

    Manzanares A, E.; Vega C, H.R.; Letechipia de Leon, C.; Guzman E, L.J.; Garcia T, M.

    2004-01-01

    The purpose of this study was to evaluate the effect of low gamma ray intensity upon Hsp70 expression in human Iymphocytes. The heat shock proteins (Hsp) family, are a group of proteins present in all living organism, therefore there are highly conserved and are related to adaptation and evolution. At cellular level these proteins acts as chaperones correcting denatured proteins. When a stress agent, such heavy metals, UV, heat, etc. is affecting a cell a response to this aggression is triggered only through over expression of Hsp. Several studies has been carried out in which the cellular effect are observed, mostly of these studies uses large doses, but very few studies are related with low doses. Blood of healthy volunteers was obtained and the Iymphocytes were isolated by ficoll- histopaque gradient. Experimental lots were irradiated in a 137 Cs gamma-ray. Hsp70 expression was found since 0.5 c Gy, indicating a threshold to very low doses of gamma rays. (Author)

  9. Cytosolic Hsp70/Hsc70 protein expression in lymphocytes exposed to low gamma-ray dose

    Energy Technology Data Exchange (ETDEWEB)

    Manzanares A, E.; Vega C, H.R.; Letechipia de Leon, C. [Unidades Academicas de Estudios Nucleares, UAZ, A.P. 336, 98000 Zacatecas (Mexico)]. E-mail: emanz@cantera.reduaz.mx; Guzman E, L.J. [Unidad Academica de Biologia Experimental, Guadalupe, Zacatecas (Mexico); Garcia T, M. [LIBRA, Centro I and D, Campus Miguel Delibes, Valladolid 47011 (Spain)

    2004-07-01

    The purpose of this study was to evaluate the effect of low gamma ray intensity upon Hsp70 expression in human Iymphocytes. The heat shock proteins (Hsp) family, are a group of proteins present in all living organism, therefore there are highly conserved and are related to adaptation and evolution. At cellular level these proteins acts as chaperones correcting denatured proteins. When a stress agent, such heavy metals, UV, heat, etc. is affecting a cell a response to this aggression is triggered only through over expression of Hsp. Several studies has been carried out in which the cellular effect are observed, mostly of these studies uses large doses, but very few studies are related with low doses. Blood of healthy volunteers was obtained and the Iymphocytes were isolated by ficoll- histopaque gradient. Experimental lots were irradiated in a {sup 137} Cs gamma-ray. Hsp70 expression was found since 0.5 c Gy, indicating a threshold to very low doses of gamma rays. (Author)

  10. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    Science.gov (United States)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  11. Involvement of p27CIP/KIP in HSP25 or HSP70 Mediated Adaptive Response by Low Dose Radiation

    International Nuclear Information System (INIS)

    Seo, Hang Rhan; Lee, Yoon Jin; Lee, Su Jae; Bae, Sang woo; Lee, Yun Sil

    2005-01-01

    Adaptive responses that reduce the harmful effects of subsequent exposure to high-dose radiation have demonstrated in chromosome aberration, cell survival, sister chromatid exchanges, micronucleus induction, mutation and neoplastic transformation. The mechanisms and conditions for the adaptive response to radiation have not been clarified, although the continuous production of free radicals from radiation and other sources has stimulated cells to evolve a repair system for chromosome breaks. An alteration of the DNA molecule triggers the repair system, and frequent activation may increase the general repair capacity, irrespective of the cause of the damage. Besides, cell cycle regulation systems, antioxidant defense systems, molecular chaperone or stress-response systems. Our previous data showed that when cells were preirradiated with 1cGy, they showed the adaptive response. A reduction of apoptosis by low-dose preirradiation is another potential mechanism for this effect. We previously demonstrated that mouse RIF cells, which did not induce HSP25 and HSP70 did not exhibit a adaptive response after 1cGy preirradiation. whereas the thermoresistant TR cells, which expressed inducible HSP25 and HSP70 showed a response. Moreover, when HSP70 and HSP25 were transfected to RIF cells, the cells acquired adaptive response. In this study, to elucidate the mechanisms in induction of adaptiveresponse, we compared cell cycle distribution by low dose radiation after HSP25 or HSP70 transfected cells and p27CIP/KIP is responsible for the different induction of adaptive response

  12. Exercise Training under Exposure to Low Levels of Fine Particulate Matter: Effects on Heart Oxidative Stress and Extra-to-Intracellular HSP70 Ratio

    Directory of Open Access Journals (Sweden)

    Aline Sfalcin Mai

    2017-01-01

    Full Text Available Fine particulate matter (PM2.5 promotes heart oxidative stress (OS and evokes anti-inflammatory responses observed by increased intracellular 70 kDa heat shock proteins (iHSP70. Furthermore, PM2.5 increases the levels of these proteins in extracellular fluids (eHSP70, which have proinflammatory roles. We investigated whether moderate and high intensity training under exposure to low levels of PM2.5 modifies heart OS and the eHSP70 to iHSP70 ratio (H-index, a biomarker of inflammatory status. Male mice (n=32, 30 days old, were divided into six groups for 12 weeks: control (CON, moderate (MIT and high intensity training (HIT, exposure to 5 μg of PM2.5 daily (PM2.5, and moderate and high intensity training exposed to PM2.5 (MIT + PM2.5 and HIT + PM2.5 groups. The CON and PM2.5 groups remained sedentary. The MIT + PM2.5 group showed higher heart lipid peroxidation levels than the MIT and PM2.5 groups. HIT and HIT + PM2.5 showed higher heart lipid peroxidation levels and lower eHSP70 and H-index levels compared to sedentary animals. No alterations were found in heart antioxidant enzyme activity or iHSP70 levels. Moderate exercise training under exposure to low levels of PM2.5 induces heart OS but does not modify eHSP70 to iHSP70 ratio (H-index. High intensity exercise training promotes anti-inflammatory profile despite exposure to low levels of PM2.5.

  13. An RNA aptamer specific to Hsp70-ATP conformation inhibits its ATPase activity independent of Hsp40.

    Science.gov (United States)

    Thirunavukarasu, Deepak; Shi, Hua

    2015-04-01

    The highly conserved and ubiquitous molecular chaperone heat shock protein 70 (Hsp70) plays a critical role in protein homeostasis (proteostasis). Controlled by its ATPase activity, Hsp70 cycles between two conformations, Hsp70-ATP and Hsp70-ADP, to bind and release its substrate. Chemical tools with distinct modes of action, especially those capable of modulating the ATPase activity of Hsp70, are being actively sought after in the mechanistic dissection of this system. Here, we report a conformation-specific RNA aptamer that binds only to Hsp70-ATP but not to Hsp70-ADP. We have refined this aptamer and demonstrated its inhibitory effect on Hsp70's ATPase activity. We have also shown that this inhibitory effect on Hsp70 is independent of its interaction with the Hsp40 co-chaperone. As Hsp70 is increasingly being recognized as a drug target in a number of age related diseases such as neurodegenerative, protein misfolding diseases and cancer, this aptamer is potentially useful in therapeutic applications. Moreover, this work also demonstrates the feasibility of using aptamers to target ATPase activity as a general therapeutic strategy.

  14. Hsp70/Hsp90 organising protein (hop): beyond interactions with chaperones and prion proteins.

    Science.gov (United States)

    Baindur-Hudson, Swati; Edkins, Adrienne L; Blatch, Gregory L

    2015-01-01

    The Hsp70/Hsp90 organising protein (Hop), also known as stress-inducible protein 1 (STI1), has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins. Consequently, Hop is implicated in a number of key signalling pathways, including aberrant pathways leading to cancer. However, Hop is also secreted and it is now well established that Hop also serves as a receptor for the prion protein, PrP(C). The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrP(C). While Hop has been shown to have various cellular functions, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseases states.

  15. The Complex Function of Hsp70 in Metastatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Juhasz, Kata; Lipp, Anna-Maria; Nimmervoll, Benedikt; Sonnleitner, Alois; Hesse, Jan; Haselgruebler, Thomas; Balogi, Zsolt, E-mail: zsolt.balogi@cbl.at [Center for Advanced Bioanalysis GmbH, Gruberstr. 40-42, A-4020 Linz (Austria)

    2013-12-20

    Elevated expression of the inducible heat shock protein 70 (Hsp70) is known to correlate with poor prognosis in many cancers. Hsp70 confers survival advantage as well as resistance to chemotherapeutic agents, and promotes tumor cell invasion. At the same time, tumor-derived extracellular Hsp70 has been recognized as a “chaperokine”, activating antitumor immunity. In this review we discuss localization dependent functions of Hsp70 in the context of invasive cancer. Understanding the molecular principles of metastasis formation steps, as well as interactions of the tumor cells with the microenvironment and the immune system is essential for fighting metastatic cancer. Although Hsp70 has been implicated in different steps of the metastatic process, the exact mechanisms of its action remain to be explored. Known and potential functions of Hsp70 in controlling or modulating of invasion and metastasis are discussed.

  16. Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

    Science.gov (United States)

    Kisliouk, Tatiana; Cramer, Tomer; Meiri, Noam

    2017-05-01

    Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress. © 2017 International Society for Neurochemistry.

  17. Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae.

    Science.gov (United States)

    de Groot, E; Bebelman, J P; Mager, W H; Planta, R J

    2000-02-01

    Changing the growth mode of Saccharomyces cerevisiae by adding fermentable amounts of glucose to cells growing on a non-fermentable carbon source leads to rapid repression of general stress-responsive genes like HSP12. Remarkably, glucose repression of HSP12 appeared to occur even at very low glucose concentrations, down to 0.005%. Although these low levels of glucose do not induce fermentative growth, they do act as a growth signal, since upon addition of glucose to a concentration of 0.02%, growth rate increased and ribosomal protein gene transcription was up-regulated. In an attempt to elucidate how this type of glucose signalling may operate, several signalling mutants were examined. Consistent with the low amounts of glucose that elicit HSP12 repression, neither the main glucose-repression pathway nor cAMP-dependent activation of protein kinase A appeared to play a role in this regulation. Using mutants involved in glucose metabolism, evidence was obtained suggesting that glucose 6-phosphate serves as a signalling molecule. To identify the target for glucose repression on the promoter of the HSP12 gene, a promoter deletion series was used. The major transcription factors governing (stress-induced) transcriptional activation of HSP12 are Msn2p and Msn4p, binding to the general stress-responsive promoter elements (STREs). Surprisingly, glucose repression of HSP12 appeared to be independent of Msn2/4p: HSP12 transcription in glycerol-grown cells was unaffected in a deltamsn2deltamsn4 strain. Nevertheless, evidence was obtained that STRE-mediated transcription is the target of repression by low amounts of glucose. These data suggest that an as yet unidentified factor is involved in STRE-mediated transcriptional regulation of HSP12.

  18. Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

    Science.gov (United States)

    Bernardo, Bianca C; Sapra, Geeta; Patterson, Natalie L; Cemerlang, Nelly; Kiriazis, Helen; Ueyama, Tomomi; Febbraio, Mark A; McMullen, Julie R

    2015-01-01

    Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

  19. Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation.

    Directory of Open Access Journals (Sweden)

    Bianca C Bernardo

    Full Text Available Previous animal studies had shown that increasing heat shock protein 70 (Hsp70 using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF. AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC. Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.

  20. Stress, and pathogen response gene expression in modeled microgravity

    Science.gov (United States)

    Sundaresan, Alamelu; Pellis, Neal R.

    2006-01-01

    Purpose: Immune suppression in microgravity has been well documented. With the advent of human exploration and long-term space travel, the immune system of the astronaut must be optimally maintained. It is important to investigate the expression patterns of cytokine genes, because they are directly related to immune response. Heat shock proteins (HSPs), also called stress proteins, are a group of proteins that are present in the cells of every life form. These proteins are induced when a cell responds to stressors such as heat, cold and oxygen deprivation. Microgravity is another stressor that may regulate HSPs. Heat shock proteins trigger immune response through activities that occur both inside the cell (intracellular) and outside the cell (extracellular). Knowledge about these two gene groups could lead to establishment of a blueprint of the immune response and adaptation-related genes in the microgravity environment. Methods: Human peripheral blood cells were cultured in 1g (T flask) and modeled microgravity (MMG, rotating-wall vessel) for 24 and 72 hours. Cell samples were collected and subjected to gene array analysis using the Affymetrix HG_U95 array. Data was collected and subjected to a two-way analysis of variance. The genes related to immune and stress responses were analyzed. Results and Conclusions: HSP70 was up-regulated by more than two fold in microgravity culture, while HSP90 was significantly down-regulated. HSP70 is not typically expressed in all kinds of cells, but it is expressed at high levels in stress conditions. HSP70 participates in translation, protein translocation, proteolysis and protein folding, suppressing aggregation and reactivating denatured proteins. Increased serum HSP70 levels correlate with a better outcome for heat-stroke or severe trauma patients. At the same time, elevated serum levels of HSP70 have been detected in patients with peripheral or renal vascular disease. HSP90 has been identified in the cytosol, nucleus and

  1. Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

    DEFF Research Database (Denmark)

    Singh, Ripudaman; Kølvraa, Steen; Bross, Peter

    2006-01-01

    Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR...... was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible...

  2. Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

    Science.gov (United States)

    Rohmer, Stanimira; Mainka, Astrid; Knippertz, Ilka; Hesse, Andrea; Nettelbeck, Dirk M

    2008-04-01

    Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors. (c) 2008 John Wiley & Sons, Ltd.

  3. Cadmium tolerance in seven Daphnia magna clones is associated with reduced hsp70 baseline levels and induction

    International Nuclear Information System (INIS)

    Haap, Timo; Koehler, Heinz-R.

    2009-01-01

    The stress protein hsp70 is part of the intracellular alarm and repair system which enables organisms to counteract negative effects of toxicants on protein integrity. Under long-term selection pressure exerted by environmental pollution, in particular heavy metals, this system may be expected to play a major role in the course of local, microevolutionary events leading to the acquisition of toxicant resistance. Seven clones of Daphnia magna from different geographical regions were characterized regarding their sensitivity to Cd, their hsp70 expression, and Cd accumulation. In an acute immobilisation assay, the tested clones showed remarkable differences in their sensitivity to Cd. The highest EC 50 values by far were obtained for the clone displaying lowest hsp70 expression. In general, hsp70 levels reflected the order of sensitivity to Cd among the seven clones reciprocally. Clonal variations in sensitivity and hsp70 expression could not be related to differential accumulation of Cd, though. In summary, the association of stress insensitivity with low hsp70 induction which has been exemplarily reported for populations of different invertebrates under strong selection pressure could be affirmed for a largely parthenogenetic species for the first time. Furthermore, our observation has serious consequences for the interpretation of toxicological assays using a single D. magna clone solely.

  4. The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

    Directory of Open Access Journals (Sweden)

    Michael O Daniyan

    Full Text Available Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1 or a human Hsp70 (HSPA1A, indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

  5. The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider; Lange, Marianne; Ahring, Birgitte Kiær

    1999-01-01

    The hsp70 (dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic...... thermoautotrophicum Delta H, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the Delta H proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion-by comparison with homologs from Gram...

  6. 热应激大鼠心脏损伤与血淋巴细胞热休克蛋白70的关系%Relationship between heat stress-induced cardiomyocyte injury and HSP70 in lymphocytes in rats

    Institute of Scientific and Technical Information of China (English)

    井然; 钱捷; 弓景波; 梅竹松; 王新兴; 钱令嘉

    2012-01-01

    目的 观察不同高温环境中热应激大鼠心肌损伤状况,探索血液淋巴细胞中HSP70水平与心肌损伤的相关性规律及其意义.方法 50只雄性Wistar大鼠随机分为常温(22~25℃)组,36、37、38、39 cC热应激组.以人工高温舱对大鼠进行热暴露.采用电子肛温计检测大鼠肛温,采用八导生理记录仪观测大鼠心电图的变化,采用酶学试剂盒检测大鼠血清乳酸脱氢酶(LDH)和肌酸激酶同工酶(CK-MB)活力,采用Western Blot方法对大鼠外周血淋巴细胞热休克蛋白70(HSP70)含量进行半定量分析.结果 与常温组比较,36~39℃组体温均升高(均P<0.05);37~39℃组Q-T间期延长和R波振幅下降(均P<0.05);38~39℃组血清中LDH和CK-MB活力升高(均P<0.05).淋巴细胞中HSP70表达水平随环境温度的变化呈现不同的变化规律,热环境温度≤37℃时,HSP70表达随气温升高而显著增加(均P<0.05),热环境温度≥38℃后,HSP70表达随气温升高而下降,但仍显著高于常温组(均P<0.05).结论 热应激可导致大鼠心肌损伤和外周血淋巴细胞HSP70表达水平发生规律性变化,提示HSP70可能是热应激致心肌损伤的一种生物标志分子,在高温所致心肌损伤发生中具有重要作用.%Objective To investigate the myocardium damage of rats induced by hot environment and its correlation with HSP70 level in blood lymphocytes. Methods Fifty Wistar rats were randomly devided into one normal temperature group and four heat stress groups (36, 37, 38, 39 ℃). The artificial chamber with high temperature was used to establish the hot environment for rats. Rectal temperature of rats was detected by electronic thermograph. The electrocardiogram was examined by eight -channel physiological recorder. The activity of LDH and CK -MB were determined with biochemical methods. The expression level of HSP70 in blood lymphocytes was analyzed by Western Blot Results Compared with normal temperature

  7. Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia.

    Science.gov (United States)

    Doberentz, Elke; Markwerth, Philipp; Wagner, Rebecca; Madea, Burkhard

    2017-09-01

    Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

  8. Differential gene expressions in testes of L2 strain Taiwan country chicken in response to acute heat stress.

    Science.gov (United States)

    Wang, Shih-Han; Cheng, Chuen-Yu; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Huang, San-Yuan

    2013-01-15

    Acute heat stress affects genes involved in spermatogenesis in mammals. However, there is apparently no elaborate research on the effects of acute heat stress on gene expression in avian testes. The purpose of this study was to investigate global gene expression in testes of the L2 strain of Taiwan country chicken after acute heat stress. Twelve roosters, 45 weeks old, were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2-hour recovery, and with 6-hour recovery, respectively. Testis samples were collected for RNA isolation and microarray analysis. Based on gene expression profiles, 169 genes were upregulated and 140 genes were downregulated after heat stress using a cutoff value of twofold or greater change. Based on gene ontology analysis, differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. A functional network analysis displayed that heat shock protein genes and related chaperones were the major upregulated groups in chicken testes after acute heat stress. A quantitative real-time polymerase chain reaction analysis of mRNA expressions of HSP70, HSP90AA1, BAG3, SERPINB2, HSP25, DNAJA4, CYP3A80, CIRBP, and TAGLN confirmed the results of the microarray analysis. Because the HSP genes (HSP25, HSP70, and HSP90AA1) and the antiapoptotic BAG3 gene were dramatically altered in heat-stressed chicken testes, we concluded that these genes were important factors in the avian testes under acute heat stress. Whether these genes could be candidate genes for thermotolerance in roosters requires further investigation. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    Directory of Open Access Journals (Sweden)

    CB Toaldo

    2001-01-01

    Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

  10. Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

    Directory of Open Access Journals (Sweden)

    Marion Hliscs

    Full Text Available Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70 family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.

  11. Role of Heat Shock Protein 70 in Induction of Stress Fiber Formation in Rat Arterial Endothelial Cells in Response to Stretch Stress

    International Nuclear Information System (INIS)

    Luo, Shan-Shun; Sugimoto, Keiji; Fujii, Sachiko; Takemasa, Tohru; Fu, Song-Bin; Yamashita, Kazuo

    2007-01-01

    We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress

  12. Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

    International Nuclear Information System (INIS)

    Geraci, Fabiana; Pinsino, Annalisa; Turturici, Guiseppina; Savona, Rosalia; Giudice, Giovanni; Sconzo, Gabriella

    2004-01-01

    Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20 h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages

  13. Hsp70-Bag3 interactions regulate cancer-related signaling networks.

    Science.gov (United States)

    Colvin, Teresa A; Gabai, Vladimir L; Gong, Jianlin; Calderwood, Stuart K; Li, Hu; Gummuluru, Suryaram; Matchuk, Olga N; Smirnova, Svetlana G; Orlova, Nina V; Zamulaeva, Irina A; Garcia-Marcos, Mikel; Li, Xiaokai; Young, Z T; Rauch, Jennifer N; Gestwicki, Jason E; Takayama, Shinichi; Sherman, Michael Y

    2014-09-01

    Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target. ©2014 American Association for Cancer Research.

  14. Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

    Science.gov (United States)

    Tsuboyama, Kotaro; Tadakuma, Hisashi; Tomari, Yukihide

    2018-05-17

    Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Hsp 70 and 90 proteins as bio indicators of stress and protein damage in human lymphocytes exposed to neutrons

    International Nuclear Information System (INIS)

    Delgado, C. E.; Letechipia de L, C.; Vega C, H. R.; Sanchez R, S. H.

    2016-09-01

    Neutrons, when interacting with the cells of the body produce free radicals, so that exposure to high doses of ionizing radiation can cause different damage to the body that can cause cell death, therefore, these effects will depend on the amount of dose, time and individual factors such as gender, age, health status and nutrition. Therefore, knowledge of cellular responses to radiation exposure is critical for developing predictive markers useful for assessing people's exposure to radiation. The purpose of this study was to estimate the cellular protein damage through the Hsp 70 and 90 proteins exposed to neutrons in human lymphocytes from clinically healthy subjects. The cell tissue was obtained by venipuncture, the lymphocytes were separated by Ficoll-Paque concentration gradient, the experimental batches were formed, thus having 5 duplicate samples, subjected to neutron irradiation in a "2"4"2Am-Be at doses of 0.25, 0.50, 0.75, 1 and 1.25 μGy at three distances 20, 21.5 and 23 cm. As a positive control, a sample exposed to heat (40 degrees Celsius) was used for 40 min. The proteins of the experimental batch were analyzed by Western-Blot and protein quantification was analyzed by densitometry, on the other hand the oxidative stress was quantified by Oxi-Blot. Was found that the neutrons at doses of 0.25 and 0.5 μGy over expressed the Hsp-70 proteins, but for Hsp-90 no over-dose expressed, there was no protein damage at the exposure doses that were established. It can be estimated that Hsp-70 proteins can serve as bio indicators of cell stress by exposure doses of 0.25 and 0.5 μGy of neutrons. (Author)

  16. Heat adaptation from regular hot water immersion decreases proinflammatory responses, HSP70 expression, and physical heat stress.

    Science.gov (United States)

    Yang, Fwu-Lin; Lee, Chia-Chi; Subeq, Yi-Maun; Lee, Chung-Jen; Ke, Chun-Yen; Lee, Ru-Ping

    2017-10-01

    Hot-water immersion (HWI) is a type of thermal therapy for treating various diseases. In our study, the physiological responses to occasional and regular HWI have been explored. The rats were divided into a control group, occasional group (1D), and regular group (7D). The 1D and 7D groups received 42°C during 15mins HWI for 1 and 7 days, respectively. The blood samples were collected for proinflammatory cytokines examinations, the heart, liver and kidney were excised for subsequent IHC analysis to measure the level of heat shock protein 70 (HSP70). The results revealed that the body temperature increased significantly during HWI on Day 3 and significantly declined on Days 6 and 7. For the 7D group, body weight, heart rate, hematocrit, platelet, osmolarity, and lactate level were lower than those in the 1D group. Furthermore, the levels of granulocyte counts, tumor necrosis factor-α, and interleukin-6 were lower in the 7D group than in the 1D group. The induction of HSP70 in the 1D group was higher than in the other groups. Physiological responses to occasional HWI are disadvantageous because of heat stress. However, adaptation to heat from regular HWI resulted in decreased proinflammatory responses and physical heat stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Suppression of cadmium-induced JNK/p38 activation and HSP70 family gene expression by LL-Z1640-2 in NIH3T3 cells

    International Nuclear Information System (INIS)

    Sugisawa, Nobusuke; Matsuoka, Masato; Okuno, Takeo; Igisu, Hideki

    2004-01-01

    When NIH3T3 cells were exposed to CdCl 2 , the three major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38, were phosphorylated in a time (1-9 h)- and dose (1-20 μM)-dependent manner. Treatment with a macrocyclic nonaketide compound, LL-Z1640-2 (10-100 ng/ml), suppressed the phosphorylation of MAPKs without affecting the total protein level in cells exposed to 10 μM CdCl 2 for 6 h. CdCl 2 -induced phosphorylation of c-Jun on Ser63 and that on Ser73, and resultant accumulation of total c-Jun protein were also suppressed by LL-Z1640-2 treatment. The in vitro kinase assays also showed significant inhibitory effects of LL-Z1640-2 (at 10 or 25 ng/ml) on JNK and p38 but less markedly. In contrast to JNK and p38, ERK activity was inhibited moderately only at 50 or 100 ng/ml LL-Z1640-2. On the other hand, other JNK inhibitors, SP600125 and L-JNKI1, failed to suppress CdCl 2 -induced activation of the JNK pathway. Among the mouse stress response genes upregulated in response to CdCl 2 exposure, the expressions of hsp68 (encoding for heat shock 70 kDa protein 1; Hsp70-1) and grp78 (encoding for 78 kDa glucose-regulated protein; Grp78) genes were suppressed by treatment with 25 ng/ml LL-Z1640-2. Thus, LL-Z1640-2 could suppress CdCl 2 -induced activation of JNK/p38 pathways and expression of HSP70 family genes in NIH3T3 cells. LL-Z1640-2 seems to be useful to analyze functions of toxic metal-induced JNK/p38 activation

  18. Heavy metals induce oxidative stress and trigger oxidative stress-mediated heat shock protein (hsp) modulation in the intertidal copepod Tigriopus japonicus.

    Science.gov (United States)

    Kim, Bo-Mi; Rhee, Jae-Sung; Jeong, Chang-Bum; Seo, Jung Soo; Park, Gyung Soo; Lee, Young-Mi; Lee, Jae-Seong

    2014-11-01

    Heat shock proteins (hsps) are induced by a wide range of environmental stressors including heavy metals in aquatic organisms. However, the effect of heavy metals on zooplankton at the molecular level remains still unclear. In this study, we measured the intracellular reactive oxygen species (ROS) level and the antioxidant enzyme activities for 96 h after exposure to five heavy metals: arsenic (As), cadmium (Cd), copper (Cu), silver (Ag), and zinc (Zn) in the intertidal copepod Tigriopus japonicus. Activities of the antioxidant enzymes were highly elevated in metal-exposed copepods, indicating that heavy metals can induce oxidative stress by generating ROS, and stimulate the involvement of antioxidant enzymes as cellular defense mechanisms. Subsequently, transcriptional changes in hsp gene families were further investigated in the metal-exposed groups for 96 h. The ROS level and glutathione (GSH) content were significantly increased in Ag-, As-, and Cu-exposed copepods, while they were only slightly elevated in Cd- and Zn-exposed groups. Based on the numbers of significantly modulated hsp genes and their expression levels for 96 h, we measured the effect of heavy metals to stress genes of T. japonicus in the following order: Cu > Zn > Ag > As > Cd, implying that Cu acts as a stronger oxidative stress inducer than other heavy metals. Of them, the expression of hsp20 and hsp70 genes was substantially modulated by exposure to heavy metals, indicating that these genes would provide a sensitive molecular biomarker for aquatic monitoring of heavy metal pollution. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

    International Nuclear Information System (INIS)

    Grossmann, Michael E.; Madden, Benjamin J.; Gao, Fan; Pang, Yuan-Ping; Carpenter, John E.; McCormick, Daniel; Young, Charles Y.F.

    2004-01-01

    Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed

  20. Accumulation of hepatic Hsp70 and plasma cortisol in Oreochromis ...

    African Journals Online (AJOL)

    The hepatic isoforms Hsp70, Hsp74 and Hsp76 were identified and quantified from copper exposures. Long-term DDT exposure did not result in significant induction of hepatic Hsp70. An increase in plasma cortisol concentration was associated with a decrease in heat shock protein accumulation after cadmium exposure, ...

  1. Hsp 70 and 90 proteins as bio indicators of stress and protein damage in human lymphocytes exposed to neutrons; Proteinas Hsp 70 y 90 como bioindicadores de estres y dano proteico en linfocitos humanos expuestos a neutrones

    Energy Technology Data Exchange (ETDEWEB)

    Delgado, C. E.; Letechipia de L, C.; Vega C, H. R.; Sanchez R, S. H., E-mail: rmfranccesco00223@hotmail.com [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Cipres No. 10, Fracc. La Penuela, 98068 Zacatecas, Zac. (Mexico)

    2016-09-15

    Neutrons, when interacting with the cells of the body produce free radicals, so that exposure to high doses of ionizing radiation can cause different damage to the body that can cause cell death, therefore, these effects will depend on the amount of dose, time and individual factors such as gender, age, health status and nutrition. Therefore, knowledge of cellular responses to radiation exposure is critical for developing predictive markers useful for assessing people's exposure to radiation. The purpose of this study was to estimate the cellular protein damage through the Hsp 70 and 90 proteins exposed to neutrons in human lymphocytes from clinically healthy subjects. The cell tissue was obtained by venipuncture, the lymphocytes were separated by Ficoll-Paque concentration gradient, the experimental batches were formed, thus having 5 duplicate samples, subjected to neutron irradiation in a {sup 242}Am-Be at doses of 0.25, 0.50, 0.75, 1 and 1.25 μGy at three distances 20, 21.5 and 23 cm. As a positive control, a sample exposed to heat (40 degrees Celsius) was used for 40 min. The proteins of the experimental batch were analyzed by Western-Blot and protein quantification was analyzed by densitometry, on the other hand the oxidative stress was quantified by Oxi-Blot. Was found that the neutrons at doses of 0.25 and 0.5 μGy over expressed the Hsp-70 proteins, but for Hsp-90 no over-dose expressed, there was no protein damage at the exposure doses that were established. It can be estimated that Hsp-70 proteins can serve as bio indicators of cell stress by exposure doses of 0.25 and 0.5 μGy of neutrons. (Author)

  2. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Science.gov (United States)

    2011-01-01

    Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

  3. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Directory of Open Access Journals (Sweden)

    Serrano Carmen

    2011-01-01

    Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

  4. Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

    Science.gov (United States)

    Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

    1994-09-23

    Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

  5. Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

    Directory of Open Access Journals (Sweden)

    Mathias Gehrmann

    Full Text Available We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70 on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG, the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+ tumor cells, in vitro.The specificity of carboxy-fluorescein (CF- labeled TPP (TPP to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+ and MDA-MB-231 (75% memHsp70+ cells compared to T47D cells (29% memHsp70+ that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.This study demonstrates the specific binding and rapid

  6. Effect of electromagnetic fields at 2.45 GHz on the levels of cellular stress proteins HSP-90 and 70 in the rat thyroid

    International Nuclear Information System (INIS)

    Misa Agustino, M. J.; Alvarez-Folgueras, M.; Jorge-Mora, M. T.; Jorge Barreiro, F. J.; Ares Pena, F. J.; Lleiro, J.; Lopez Martin, M. E.

    2011-01-01

    In this study we analyzed the cellular stress levels achieved by heat shock proteins (HSP) 90 and 70 in rat thyroid tissue after exposure to radio waves in TWG experimental system. Parallel measurements of body stress in animals by rectal temperature probes allow us to determine whether there is any interaction between temperature increases and cellular stress.

  7. Differential Targeting of Hsp70 Heat Shock Proteins HSPA6 and HSPA1A with Components of a Protein Disaggregation/Refolding Machine in Differentiated Human Neuronal Cells following Thermal Stress

    Directory of Open Access Journals (Sweden)

    Ian R. Brown

    2017-04-01

    Full Text Available Heat shock proteins (Hsps co-operate in multi-protein machines that counter protein misfolding and aggregation and involve DNAJ (Hsp40, HSPA (Hsp70, and HSPH (Hsp105α. The HSPA family is a multigene family composed of inducible and constitutively expressed members. Inducible HSPA6 (Hsp70B' is found in the human genome but not in the genomes of mouse and rat. To advance knowledge of this little studied HSPA member, the targeting of HSPA6 to stress-sensitive neuronal sites with components of a disaggregation/refolding machine was investigated following thermal stress. HSPA6 targeted the periphery of nuclear speckles (perispeckles that have been characterized as sites of transcription. However, HSPA6 did not co-localize at perispeckles with DNAJB1 (Hsp40-1 or HSPH1 (Hsp105α. At 3 h after heat shock, HSPA6 co-localized with these members of the disaggregation/refolding machine at the granular component (GC of the nucleolus. Inducible HSPA1A (Hsp70-1 and constitutively expressed HSPA8 (Hsc70 co-localized at nuclear speckles with components of the machine immediately after heat shock, and at the GC layer of the nucleolus at 1 h with DNAJA1 and BAG-1. These results suggest that HSPA6 exhibits targeting features that are not apparent for HSPA1A and HSPA8.

  8. Recombinant TgHSP70 Immunization Protects against Toxoplasma gondii Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression

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    Neide M. Silva

    2017-04-01

    Full Text Available Toxoplasma gondii is known to cause congenital infection in humans and animals and severe disease in immunocompromised individuals; consequently development of vaccines against the parasite is highly necessary. Under stress conditions, T. gondii expresses the highly immunogenic heat shock protein 70 (TgHSP70. Here, we assessed the protective efficacy of rTgHSP70 immunization combined with Alum in oral ME-49 T. gondii infection and the mechanisms involved on it. It was observed that immunized mice with rTgHSP70 or rTgHSP70 adsorbed in Alum presented a significantly reduced number of cysts in the brain that was associated with increased iNOS+ cell numbers in the organ, irrespective the use of the adjuvant. Indeed, ex vivo experiments showed that peritoneal macrophages pre-stimulated with rTgHSP70 presented increased NO production and enhanced parasite killing, and the protein was able to directly stimulate B cells toward antibody producing profile. In addition, rTgHSP70 immunization leads to high specific antibody titters systemically and a mixed IgG1/IgG2a response, with predominance of IgG1 production. Nonetheless, it was observed that the pretreatment of the parasite with rTgHSP70 immune sera was not able to control T. gondii internalization and replication by NIH fibroblast neither peritoneal murine macrophages, nor anti-rTgHSP70 antibodies were able to kill T. gondii by complement-mediated lysis, suggesting that these mechanisms are not crucial to resistance. Interestingly, when in combination with Alum, rTgHSP70 immunization was able to reduce inflammation in the brain of infected mice and in parallel anti-rTgHSP70 immune complexes in the serum. In conclusion, immunization with rTgHSP70 induces massive amounts of iNOS expression and reduced brain parasitism, suggesting that iNOS expression and consequently NO production in the brain is a protective mechanism induced by TgHSP70 immunization, therefore rTgHSP70 can be a good candidate for

  9. Andrographolide induces degradation of mutant p53 via activation of Hsp70.

    Science.gov (United States)

    Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

    2018-05-22

    The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

  10. Survival rate and expression of Heat-shock protein 70 and Frost genes after temperature stress in Drosophila melanogaster lines that are selected for recovery time from temperature coma.

    Science.gov (United States)

    Udaka, Hiroko; Ueda, Chiaki; Goto, Shin G

    2010-12-01

    In this study, we investigated the physiological mechanisms underlying temperature tolerance using Drosophila melanogaster lines with rapid, intermediate, or slow recovery from heat or chill coma that were established by artificial selection or by free recombination without selection. Specifically, we focused on the relationships among their recovery from heat or chill coma, survival after severe heat or cold, and survival enhanced by rapid cold hardening (RCH) or heat hardening. The recovery time from heat coma was not related to the survival rate after severe heat. The line with rapid recovery from chill coma showed a higher survival rate after severe cold exposure, and therefore the same mechanisms are likely to underlie these phenotypes. The recovery time from chill coma and survival rate after severe cold were unrelated to RCH-enhanced survival. We also examined the expression of two genes, Heat-shock protein 70 (Hsp70) and Frost, in these lines to understand the contribution of these stress-inducible genes to intraspecific variation in recovery from temperature coma. The line showing rapid recovery from heat coma did not exhibit higher expression of Hsp70 and Frost. In addition, Hsp70 and Frost transcription levels were not correlated with the recovery time from chill coma. Thus, Hsp70 and Frost transcriptional regulation was not involved in the intraspecific variation in recovery from temperature coma. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Cytosine deletion at AP2-box region of HSP70 promoter and its ...

    Indian Academy of Sciences (India)

    Cytosine deletion at AP2-box region of HSP70 promoter and its influence on semen quality traits in crossbred bulls ... Laboratory, ICAR-Central Institute for Research on Cattle, Meerut 250 001, India; School of Atmospheric Stress Management, ICAR-National Institute of Abiotic Stress Management, Baramati 413 115, India ...

  12. Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease

    Directory of Open Access Journals (Sweden)

    Jinlong Jian

    2016-11-01

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase. Recently it was reported that progranulin (PGRN insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

  13. Comparative baseline levels of mercury, Hsp 70 and Hsp 60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska.

    Science.gov (United States)

    Duffy, L K; Scofield, E; Rodgers, T; Patton, M; Bowyer, R T

    1999-10-01

    In subsistence fish; northern pike (Esox lucius), burbot (Lota lota), whitefish (Coregonus nelsoni), grayling (Thymallus arcticus) and sheefish (Stenodus lencichthys), we determined the Hsp 60 and Hsp 70 levels in 31 samples from adult fish gills. A dot-blot analysis using antibodies to either Hsp 70 or Hsp 60 showed the average Hsp 70 concentration was 9.1 microg/mg protein, while the average Hsp 60 concentration was 147.4 microg/mg protein. Mercury levels in muscle tissue in these fish averaged 0.382 ppm. Using a subset of samples (n = 24), we determined that the major component in the muscle of Alaskan subsistence fish was methyl mercury. No correlation was observed between Hsp 60 or Hsp 70 expression in gill tissue and mercury concentrations in muscle tissue. Hsp 60 and Hsp 70 protein levels in the gills were correlated.

  14. Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

    NARCIS (Netherlands)

    Michels, AA; Kanon, B; Bensaude, O; Kampinga, HH

    1999-01-01

    Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272,

  15. The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle.

    Science.gov (United States)

    Cobb, David W; Florentin, Anat; Fierro, Manuel A; Krakowiak, Michelle; Moore, Julie M; Muralidharan, Vasant

    2017-01-01

    Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum . IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is

  16. Hsf and Hsp gene families in Populus: genome-wide identification, organization and correlated expression during development and in stress responses.

    Science.gov (United States)

    Zhang, Jin; Liu, Bobin; Li, Jianbo; Zhang, Li; Wang, Yan; Zheng, Huanquan; Lu, Mengzhu; Chen, Jun

    2015-03-14

    Heat shock proteins (Hsps) are molecular chaperones that are involved in many normal cellular processes and stress responses, and heat shock factors (Hsfs) are the transcriptional activators of Hsps. Hsfs and Hsps are widely coordinated in various biological processes. Although the roles of Hsfs and Hsps in stress responses have been well characterized in Arabidopsis, their roles in perennial woody species undergoing various environmental stresses remain unclear. Here, a comprehensive identification and analysis of Hsf and Hsp families in poplars is presented. In Populus trichocarpa, we identified 42 paralogous pairs, 66.7% resulting from a whole genome duplication. The gene structure and motif composition are relatively conserved in each subfamily. Microarray and quantitative real-time RT-PCR analyses showed that most of the Populus Hsf and Hsp genes are differentially expressed upon exposure to various stresses. A coexpression network between Populus Hsf and Hsp genes was generated based on their expression. Coordinated relationships were validated by transient overexpression and subsequent qPCR analyses. The comprehensive analysis indicates that different sets of PtHsps are downstream of particular PtHsfs and provides a basis for functional studies aimed at revealing the roles of these families in poplar development and stress responses.

  17. Sequence analysis of the 3’-untranslated region of HSP70 (type I genes in the genus Leishmania: its usefulness as a molecular marker for species identification

    Directory of Open Access Journals (Sweden)

    Requena Jose M

    2012-04-01

    Full Text Available Abstract Background The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods In the present study, we analyzed the 3’-untranslated region (UTR of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3´-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.

  18. Plasmodium falciparum Hop (PfHop Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity.

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    Tawanda Zininga

    Full Text Available Heat shock proteins (Hsps play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70 is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90 facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop. We previously characterised the Hop protein from Plasmodium falciparum (PfHop. We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we

  19. Conserved effects of salinity acclimation on thermal tolerance and hsp70 expression in divergent populations of threespine stickleback (Gasterosteus aculeatus).

    Science.gov (United States)

    Metzger, David C H; Healy, Timothy M; Schulte, Patricia M

    2016-10-01

    In natural environments, organisms must cope with complex combinations of abiotic stressors. Here, we use threespine stickleback (Gasterosteus aculeatus) to examine how changes in salinity affect tolerance of high temperatures. Threespine stickleback inhabit a range of environments that vary in both salinity and thermal stability making this species an excellent system for investigating interacting stressors. We examined the effects of environmental salinity on maximum thermal tolerance (CTMax) and 70 kDa heat shock protein (hsp70) gene expression using divergent stickleback ecotypes from marine and freshwater habitats. In both ecotypes, the CTMax of fish acclimated to 20 ppt was significantly higher compared to fish acclimated to 2 ppt. The effect of salinity acclimation on the expression of hsp70-1 and hsp70-2 was similar in both the marine and freshwater stickleback ecotype. There were differences in the expression profiles of hsp70-1 and hsp70-2 during heat shock, with hsp70-2 being induced earlier and to a higher level compared to hsp70-1. These data suggest that the two hsp70 isoforms may have functionally different roles in the heat shock response. Lastly, acute salinity challenge coupled with heat shock revealed that the osmoregulatory demands experienced during the heat shock response have a larger effect on the hsp70 expression profile than does the acclimation salinity.

  20. Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

    Science.gov (United States)

    Heinen, R C; Diniz-Mendes, L; Silva, J T; Paschoalin, V M F

    2006-11-01

    Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

  1. Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

    Directory of Open Access Journals (Sweden)

    R.C. Heinen

    2006-11-01

    Full Text Available Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

  2. Regulation of human heme oxygenase-1 gene expression under thermal stress.

    Science.gov (United States)

    Okinaga, S; Takahashi, K; Takeda, K; Yoshizawa, M; Fujita, H; Sasaki, H; Shibahara, S

    1996-06-15

    Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

  3. Heritability of hsp70 expression in the beetle Tenebrio molitor: Ontogenetic and environmental effects.

    Science.gov (United States)

    Lardies, Marco A; Arias, María Belén; Poupin, María Josefina; Bacigalupe, Leonardo D

    2014-08-01

    Ectotherms constitute the vast majority of terrestrial biodiversity and are especially likely to be vulnerable to climate warming because their basic physiological functions such as locomotion, growth, and reproduction are strongly influenced by environmental temperature. An integrated view about the effects of global warming will be reached not just establishing how the increase in mean temperature impacts the natural populations but also establishing the effects of the increase in temperature variance. One of the molecular responses that are activated in a cell under a temperature stress is the heat shock protein response (HSP). Some studies that have detected consistent differences among thermal treatments and ontogenetic stages in HSP70 expression have assumed that these differences had a genetic basis and consequently expression would be heritable. We tested for changes in quantitative genetic parameters of HSP70 expression in a half-sib design where individuals of the beetle Tenebrio molitor were maintained in constant and varying thermal environments. We estimated heritability of HSP70 expression using a linear mixed modelling approach in different ontogenetic stages. Expression levels of HSP70 were consistently higher in the variable environment and heritability estimates were low to moderate. The results imply that within each ontogenetic stage additive genetic variance was higher in the variable environment and in adults compared with constant environment and larvae stage, respectively. We found that almost all the genetic correlations across ontogenetic stages and environment were positive. These suggest that directional selection for higher levels of expression in one environment will result in higher expression levels of HSP70 on the other environment for the same ontogenetic stage. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70.

    Science.gov (United States)

    Colvin, Teresa A; Gabai, Vladimir L; Sherman, Michael Y

    2014-01-01

    Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

  5. Critical analysis of eukaryotic phylogeny: a case study based on the HSP70 family.

    Science.gov (United States)

    Germot, A; Philippe, H

    1999-01-01

    Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species; and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes, likely after the mitochondrial endosymbiosis.

  6. An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats.

    Science.gov (United States)

    Wang, Yu; Nie, Huaming; Gu, Xiaobin; Wang, Tao; Huang, Xing; Chen, Lin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

    2015-09-15

    Infections with the tapeworm Taenia multiceps are problematic for ruminant farming worldwide. Here we develop a novel and rapid method for serodiagnosis of T. multiceps infections via an indirect ELISA (iELISA) that uses a heat shock protein, namely, TmHSP70. We extracted the total RNA of T. multiceps from the protoscoleces of cysts dissected from the brains of infected goats. Subsequently, we successfully amplified, cloned and expressed the TmHSP70 gene in Escherichia coli BL21 (DE3). Western blot analysis showed that the recombinant protein (∼34 kDa molecular weight) was recognized by the coenurosis positive serum. Given these initial, robust immunogenic properties for recombinant TmHSP protein, we assessed the ELISA-based serodiagnostic potential of this gene. The indirect ELISA was then optimized to 2.70 μg/well dilution for antigen and 1:80 dilution for serum,while the cut-off value is 0.446. We report that our novel TmHSP ELISA detected T. multiceps sera with a sensitivity of 1:10240 and a specificity of 83.3% (5/6). In a preliminary application, this assay correctly confirmed T. multiceps infection in 30 infected goats, consistent with the clinical examination. This study has revealed that our novel iELISA, which uses the rTmHSP protein, provides a rapid test for diagnosing coenurosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70 Function

    Directory of Open Access Journals (Sweden)

    Tawanda Zininga

    2017-07-01

    Full Text Available Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70 function. Heat shock protein 70 (Hsp70 are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function.

  8. Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease.

    Science.gov (United States)

    Jian, Jinlong; Tian, Qing-Yun; Hettinghouse, Aubryanna; Zhao, Shuai; Liu, Helen; Wei, Jianlu; Grunig, Gabriele; Zhang, Wujuan; Setchell, Kenneth D R; Sun, Ying; Overkleeft, Herman S; Chan, Gerald L; Liu, Chuan-Ju

    2016-11-01

    Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

    Science.gov (United States)

    Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

    2017-10-01

    By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

  10. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

    Science.gov (United States)

    Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

    2009-10-12

    Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

  11. Acclimation-dependent expression of heat shock protein 70 in Pacific abalone ( Haliotis discus hannai Ino) and its acute response to thermal exposure

    Science.gov (United States)

    Li, Jiaqi; He, Qingguo; Sun, Hui; Liu, Xiao

    2012-01-01

    Heat shock protein 70 (Hsp70) is one important member of heat shock protein (Hsp) family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone ( Haliotis discus hannai Ino). For the chronic exposure, abalones were maintained at 8, 12, 20, and 30°C for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30°C-acclimated group, followed by the 8°C-acclimated group and then the 12°C- and 20°C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8°C and 38°C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

  12. Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity.

    Science.gov (United States)

    Sung, Y Y; Roberts, R J; Bossier, P

    2012-08-01

    Exposure to TEX-OE®, a patented extract of the prickly pear cactus (Opuntia ficus indica) containing chaperone-stimulating factor, was shown to protect common carp, Cyprinus carpio L., fingerlings against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to 5.92 mg L(-1) NH(3) , a level determined in the ammonia challenge bioassay as the 1-h LD50 concentration for this species. Survival of TEX-OE®-pre-exposed fish was enhanced by 20% over non-exposed controls during lethal ammonia challenge (14.21 mg L(-1)  NH(3) ). Increase in the levels of gill and muscle Hsp70 was evident in TEX-OE®-pre-exposed fish but not in the unexposed controls, indicating that application of TEX-OE® accelerated carp endogenous Hsp70 synthesis during ammonia perturbation. Protection against ammonia was correlated with Hsp70 accretion. © 2012 Blackwell Publishing Ltd.

  13. Effect of electromagnetic fields at 2.45 GHz on the levels of cellular stress proteins HSP-90 and 70 in the rat thyroid; Efecto de los campos electromagneticos a 2,45 GHz sobre los niveles de proteinas de estres celular HSP-90 y 70 en el toroides de rata

    Energy Technology Data Exchange (ETDEWEB)

    Misa Agustino, M. J.; Alvarez-Folgueras, M.; Jorge-Mora, M. T.; Jorge Barreiro, F. J.; Ares Pena, F. J.; Lleiro, J.; Lopez Martin, M. E.

    2011-07-01

    In this study we analyzed the cellular stress levels achieved by heat shock proteins (HSP) 90 and 70 in rat thyroid tissue after exposure to radio waves in TWG experimental system. Parallel measurements of body stress in animals by rectal temperature probes allow us to determine whether there is any interaction between temperature increases and cellular stress.

  14. Comprehensive identification and expression analysis of Hsp90s gene family in Solanum lycopersicum.

    Science.gov (United States)

    Zai, W S; Miao, L X; Xiong, Z L; Zhang, H L; Ma, Y R; Li, Y L; Chen, Y B; Ye, S G

    2015-07-14

    Heat shock protein 90 (Hsp90) is a protein produced by plants in response to adverse environmental stresses. In this study, we identified and analyzed Hsp90 gene family members using a bioinformatic method based on genomic data from tomato (Solanum lycopersicum L.). The results illustrated that tomato contains at least 7 Hsp90 genes distributed on 6 chromosomes; protein lengths ranged from 267-794 amino acids. Intron numbers ranged from 2-19 in the genes. The phylogenetic tree revealed that Hsp90 genes in tomato (Solanum lycopersicum L.), rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana L.) could be divided into 5 groups, which included 3 pairs of orthologous genes and 4 pairs of paralogous genes. Expression analysis of RNA-sequence data showed that the Hsp90-1 gene was specifically expressed in mature fruits, while Hsp90-5 and Hsp90-6 showed opposite expression patterns in various tissues of cultivated and wild tomatoes. The expression levels of the Hsp90-1, Hsp90-2, and Hsp90- 3 genes in various tissues of cultivated tomatoes were high, while both the expression levels of genes Hsp90-3 and Hsp90-4 were low. Additionally, quantitative real-time polymerase chain reaction showed that these genes were involved in the responses to yellow leaf curl virus in tomato plant leaves. Our results provide a foundation for identifying the function of the Hsp90 gene in tomato.

  15. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

    Science.gov (United States)

    Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

    2017-06-07

    Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

  16. BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

    Science.gov (United States)

    Rauch, Jennifer N; Tse, Eric; Freilich, Rebecca; Mok, Sue-Ann; Makley, Leah N; Southworth, Daniel R; Gestwicki, Jason E

    2017-01-06

    Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Molecular cloning, characterization and expression of heat shock protein 70 gene from the oyster Crassostrea hongkongensis responding to thermal stress and exposure of Cu(2+) and malachite green.

    Science.gov (United States)

    Zhang, Zhanhui; Zhang, Qizhong

    2012-04-15

    Heat shock protein 70 (HSP70) acts mostly as a molecular chaperone and plays a key role in the process of protecting cells by facilitating the folding of nascent peptides and the cellular stress response. The cDNA of the oyster Crassostrea hongkongensis hsp70 (designated chhsp70) was cloned with the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full-length chhsp70 cDNA was 2251bp, consisting of a 130bp 5'-UTR, 216bp 3'-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1905bp, which encoded a polypeptide of 634 amino acids. Three classical HSP signature motifs were detected in ChHSP70, i.e., DLGTT-S-V, IFDLGGGTFDVSIL and VVLVGGSTRIPKIQK. BLAST analysis revealed that the ChHSP70 shared high identity with other bivalve HSP70. The phylogenetic analysis indicated that the ChHSP70 was a member of the HSP70 family. The chhsp70 mRNA transcripts were quantified by fluorescent real time RT-PCR under both unstressed and stressed conditions, i. e., heat shock and exposure to Cu(2+) and malachite green. Basal expression level was similar in mantle, gill, digestive gland, and heart, but higher in muscle than that in the others. A similar trend showed that the chhsp70 mRNA expression significantly increased at 3-6h, then dropped and returned to control level at 24h in the five tissues and organs mentioned above after heat shock. A clearly time-dependent expression pattern of chhsp70 mRNA in digestive gland and gill of the oyster was observed after exposure of Cu(2+) and malachite green. In the two tissues, the chhsp70 mRNA level reached the maximum at 6h after malachite green exposure and on day 4 after Cu(2+) exposure, and then decreased progressively to the control level. The results indicated that ChHSP70 of the oyster is an inducible protein, and plays an important role in response to the Cu(2+) and malachite green polluted stress, so chhsp70 might be used as a potential molecular

  18. Hsp70/J-protein machinery from Glossina morsitans morsitans, vector of African trypanosomiasis.

    Directory of Open Access Journals (Sweden)

    Stephen J Bentley

    Full Text Available Tsetse flies (Glossina spp. are the sole vectors of the protozoan parasites of the genus Trypanosoma, the causative agents of African Trypanosomiasis. Species of Glossina differ in vector competence and Glossina morsitans morsitans is associated with transmission of Trypanosoma brucei rhodesiense, which causes an acute and often fatal form of African Trypanosomiasis. Heat shock proteins are evolutionarily conserved proteins that play critical roles in proteostasis. The activity of heat shock protein 70 (Hsp70 is regulated by interactions with its J-protein (Hsp40 co-chaperones. Inhibition of these interactions are emerging as potential therapeutic targets. The assembly and annotation of the G. m. morsitans genome provided a platform to identify and characterize the Hsp70s and J-proteins, and carry out an evolutionary comparison to its well-studied eukaryotic counterparts, Drosophila melanogaster and Homo sapiens, as well as Stomoxys calcitrans, a comparator species. In our study, we identified 9 putative Hsp70 proteins and 37 putative J-proteins in G. m. morsitans. Phylogenetic analyses revealed three evolutionarily distinct groups of Hsp70s, with a closer relationship to orthologues from its blood-feeding dipteran relative Stomoxys calcitrans. G. m. morsitans also lacked the high number of heat inducible Hsp70s found in D. melanogaster. The potential localisations, functions, domain organisations and Hsp70/J-protein partnerships were also identified. A greater understanding of the heat shock 70 (Hsp70 and J-protein (Hsp40 families in G. m. morsitans could enhance our understanding of the cell biology of the tsetse fly.

  19. A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

    Science.gov (United States)

    Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

  20. 4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

    Science.gov (United States)

    Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E; Randell, Rachel L; Marando, Catherine M; Rubenstein, Ronald C

    2011-12-30

    Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

  1. The conformational dynamics of the mitochondrial Hsp70 chaperone.

    Science.gov (United States)

    Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

    2010-04-09

    Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

  2. Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4.

    Science.gov (United States)

    Ruibal, Cecilia; Castro, Alexandra; Carballo, Valentina; Szabados, László; Vidal, Sabina

    2013-11-05

    Plant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific sHsp genes. In this study we characterized the function of a sHsp in abiotic stress tolerance in the moss Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth. We identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study provides the first direct genetic

  3. Hsp70 and ceramide release by diode laser-treated mouse skin cells in vivo

    Science.gov (United States)

    Sokolovskii, G. S.; Onikienko, S. B.; Zemlyanoi, A. V.; Soboleva, K. K.; Pikhtin, N. A.; Tarasov, I. S.; Guzova, I. V.; Margulis, B. A.

    2014-12-01

    We report experimental study of generation of extracellular heat shock proteins (Hsp70) and ceramides under pulsed irradiation by quantum-well laser diodes. Our results are of great promise for applications in practical medicine such as protection against biopathogenes and abiotic stress factor challenges.

  4. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2011-07-28

    We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70\\/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

  5. Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

    Science.gov (United States)

    Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

    2016-01-01

    Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

  6. The expression and correlation of Hsp 70 and Hsp 27 in serous middle ear effusion fluids of pediatric patients-a preliminary study.

    Science.gov (United States)

    Min, Hyun Jin; Choe, Ji Won; Chang, Moon Young; Kim, Kyung Soo; Lee, Sei Young; Mun, Seog-Kyun

    2017-10-01

    Several cytokines and innate immune-associated molecules are present in middle ear effusions, but damage-associated molecular patterns (DAMPs) in middle ear effusion have not been studied. Therefore, we evaluated the role of heat shock proteins (Hsps) in the development of otitis media with effusion (OME). Serous middle ear effusions from 22 pediatric patients who were diagnosed with OME and underwent ventilation tube insertion from June 2015 to March 2017 were evaluated in our study. The levels of Hsp 90, 70, 27, IL-8, and TNF-α in effusion fluids were evaluated by enzyme-linked immunosorbent assays. The associations between the levels of these molecules and the degree of tympanic membrane inflammation were statistically evaluated. Finally, the relationships among these molecules were also evaluated. Hsp 70 and Hsp 27 were detected in all middle ear effusions, but Hsp 90 was detected in only five effusion fluid samples. IL-8 was also detected in all middle ear effusions, but TNF-α was detected in only four effusion fluid samples. When we compared the degree of tympanic membrane inflammation with the levels of Hsp 70, Hsp 27, and IL-8, which were detected in all effusion fluids, we could not find statistical significance. However, Hsp 70, Hsp 27, and IL-8 were significantly associated with each other (p effusions. Furthermore, the levels of Hsp 70 and Hsp 27 were positively correlated with each other, and were also positively associated with the neutrophil chemoattractant, IL-8. Our findings suggested that Hsp 70 and Hsp 27 might be involved in the pathophysiology of pediatric OME. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation.

    Directory of Open Access Journals (Sweden)

    Deepak Sharma

    2009-08-01

    Full Text Available The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. Hsp70s play essential roles in protein folding, transport or degradation, and are major players of cellular quality control processes. However, while several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue.We compared the ability of Ssa1p-Ssa4p from Saccharomyces cerevisiae and Ssa5p-Ssa8p from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We show that Hsp70 isoforms (i supported yeast viability yet with markedly different growth rates, (ii influenced the propagation and stability of the [PSI(+] and [URE3] prions, but iii did not significantly affect the proteasomal degradation rate of CFTR. Additionally, we show that individual Hsp70 orthologs did not induce the formation of different prion strains, but rather influenced the aggregation properties of Sup35 in vivo. Finally, we show that [URE3] curing by the overexpression of Ydj1p is Hsp70-isoform dependent.Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations. Yeast prions provide a very sensitive model to uncover this functional specialization and to explore the intricate network of chaperone/co-chaperone/substrates interactions.

  8. Expression of hsp70, hsp90 and hsf1 in the reef coral Acropora digitifera under prospective acidified conditions over the next several decades

    Directory of Open Access Journals (Sweden)

    Masako Nakamura

    2012-02-01

    Ocean acidification is an ongoing threat for marine organisms due to the increasing atmospheric CO2 concentration. Seawater acidification has a serious impact on physiologic processes in marine organisms at all life stages. On the other hand, potential tolerance to external pH changes has been reported in coral larvae. Information about the possible mechanisms underlying such tolerance responses, however, is scarce. In the present study, we examined the effects of acidified seawater on the larvae of Acropora digitifera at the molecular level. We targeted two heat shock proteins, Hsp70 and Hsp90, and a heat shock transcription factor, Hsf1, because of their importance in stress responses and in early life developmental stages. Coral larvae were maintained under the ambient and elevated CO2 conditions that are expected to occur within next 100 years, and then we evaluated the expression of hsps and hsf1 by quantitative real-time polymerase chain reaction (PCR. Expression levels of these molecules significantly differed among target genes, but they did not change significantly between CO2 conditions. These findings indicate that the expression of hsps is not changed due to external pH changes, and suggest that tolerance to acidified seawater in coral larvae may not be related to hsp expression.

  9. Plasmodium falciparum Hep1 Is Required to Prevent the Self Aggregation of PfHsp70-3.

    Directory of Open Access Journals (Sweden)

    David O Nyakundi

    Full Text Available The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70; however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1, to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria.

  10. Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy

    Directory of Open Access Journals (Sweden)

    G. Tomasello

    2011-10-01

    Full Text Available Ulcerative colitis (UC is a form of inflammatory bowel disease (IBD characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics, suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level.

  11. Conserved structure and expression of hsp70 paralogs in teleost fishes

    DEFF Research Database (Denmark)

    Metzger, David C.H.; Hansen, Jakob Hemmer; Schulte, Patricia M.

    2016-01-01

    present in the F. heteroclitus genome. Comparison of expression patterns in F. heteroclitus and Gasterosteus aculeatus demonstrates that hsp70-2 has a higher fold increase than hsp70-1 following heat shock in gill but not in muscle tissue, revealing a conserved difference in expression patterns between...

  12. Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

    International Nuclear Information System (INIS)

    Duffy, J.J.; Carper, S.W.; Gerner, E.W.

    1987-01-01

    Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

  13. Phylogenetic analysis of HSP70 and cyt b gene sequences for Chinese Leishmania isolates and ultrastructural characteristics of Chinese Leishmania sp.

    Science.gov (United States)

    Yuan, Dongmei; Qin, Hanxiao; Zhang, Jianguo; Liao, Lin; Chen, Qiwei; Chen, Dali; Chen, Jianping

    2017-02-01

    Leishmaniasis is a worldwide epidemic disease caused by the genus Leishmania, which is still endemic in the west and northwest areas of China. Some viewpoints of the traditional taxonomy of Chinese Leishmania have been challenged by recent phylogenetic researches based on different molecular markers. However, the taxonomic positions and phylogenetic relationships of Chinese Leishmania isolates remain controversial, which need for more data and further analysis. In this study, the heat shock protein 70 (HSP70) gene and cytochrome b (cyt b) gene were used for phylogenetic analysis of Chinese Leishmania isolates from patients, dogs, gerbils, and sand flies in different geographic origins. Besides, for the interesting Leishmania sp. in China, the ultrastructure of three Chinese Leishmania sp. strains (MHOM/CN/90/SC10H2, SD, GL) were observed by transmission electron microscopy. Bayesian trees from HSP70 and cyt b congruently indicated that the 14 Chinese Leishmania isolates belong to three Leishmania species including L. donovani complex, L. gerbilli, and L. (Sauroleishmania) sp. Their identity further confirmed that the undescribed Leishmania species causing visceral Leishmaniasis (VL) in China is closely related to L. tarentolae. The phylogenetic results from HSP70 also suggested the classification of subspecies within L. donovani complex: KXG-918, KXG-927, KXG-Liu, KXG-Xu, 9044, SC6, and KXG-65 belong to L. donovani; Cy, WenChuan, and 801 were proposed to be L. infantum. Through transmission electron microscopy, unexpectedly, the Golgi apparatus were not observed in SC10H2, SD, and GL, which was similar to previous reports of reptilian Leishmania. The statistical analysis of microtubule counts separated SC10H2, SD, and GL as one group from any other reference strain (L. donovani MHOM/IN/80/DD8; L. tropica MHOM/SU/74/K27; L. gerbilli MRHO/CN/60/GERBILLI). The ultrastructural characteristics of Leishmania sp. partly lend support to the phylogenetic inference that

  14. Plasmodial Hsp70s are functionally adapted to the malaria parasite life cycle

    Directory of Open Access Journals (Sweden)

    Jude M Przyborski

    2015-06-01

    Full Text Available The human malaria parasite, Plasmodium falciparum, encodes a minimal complement of six heat shock protein 70s (PfHSP70s, some of which are highly expressed and are thought to play an important role in the survival and pathology of the parasite. In addition to canonical features of molecular chaperones, these HSP70s possess properties that reflect functional adaptation to a parasitic life style, including resistance to thermal insult during fever periods and host-parasite interactions. The parasite even exports an HSP70 to the host cell where it is likely to be involved in host cell modification. This review focuses on the features of the PfHSP70s, particularly with respect to their adaptation to the malaria parasite life cycle.

  15. secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids.

    Science.gov (United States)

    De Mena, Lorena; Chhangani, Deepak; Fernandez-Funez, Pedro; Rincon-Limas, Diego E

    2017-07-03

    Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

  16. Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

    Science.gov (United States)

    Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

    2017-08-01

    In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

  17. Hsp70 Expression Profile in Preeclampsia Model of Pregnant Rat (Rattus norvegicus) after Giving the EVOO

    Science.gov (United States)

    Irianti, E.; ilyas, S.; Rosidah; Hutahaean, S.

    2017-03-01

    Heat shock protein (Hsp) has long been known to protect cells from oxidative stress. In this case an increased expression is found on several cases of preeclampsia. One of the efforts to prevent preeclampsia is by giving antioxidants such as Extra Virgin Olive Oil (EVOO) or it’s better known as olive oil (Oleoa europaea), in the form of extra virgin known for its rich antioxidant content of tocopherols (vitamin E). The purpose of this study is to determine the expression levels of Hsp70 serum on pregnant white rat model of preeclampsia after being given EVOO. This type of research is true experiment; the subjects were female white rats and male virgin with Sprague Dawley, ± 8-11 weeks old, 180g BB s / d 200g, healthy and didn’t show any physical defects. Samples were 25 animals, divided into 5 groups, which consisted of different control and treatment given to T2 (rat model of preeclampsia), T3 (rat model of preeclampsia + EVOO 0.45g/bw/day), T4 (rat model of preeclampsia + EVOO 0.9g/bw/day) and T5 (rat model of preeclampsia + EVOO 1.8g/bw/day). The determination of each group was done by simple random sampling. Result on serum levels of Hsp70 that were tested by Elisa test in rats showed the average control was 14.64 mg / ml, group T2: 22:51 mg/ml, T3: 13.62 mg/ml, T4: 15.92 mg/ml, T5: 16:09 mg/ml. ANOVA test showed the P value was 0.001 <0.005, which meant there were significant differences on serum Hsp70 levels in the control and treatment pregnant rats group. It was known that there was a significant difference level of Hsp70 serum in group of control rats with T2 (P value <0.001) after LSD test was conducted, but not so with the group T3, T4, and T5, where the difference was not significant. There was a significant difference in the levels of Hsp70 serum on group T2 and T3 (P value 0.000), T4 (0004), T5 (0000). The gift of EVOO in the treatment group which was given EVOO with even low doses was able to control the induction of Hsp70 serum levels, which

  18. Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease

    Directory of Open Access Journals (Sweden)

    Pappas Georgios J

    2008-01-01

    Full Text Available Abstract Background Citrus sudden death (CSD, a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the disease's distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV. CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms. Results A total of 286 RNA-dependent-RNA polymerase (RdRp and 284 heat shock protein 70 homolog (HSP70h gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD. Conclusion We suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence of a single CTV variant within these populations that triggers the disease.

  19. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    Science.gov (United States)

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  20. Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults

    International Nuclear Information System (INIS)

    Navarro, Anna; Faria, Melissa; Barata, Carlos; Pina, Benjamin

    2011-01-01

    Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations. - Coordinated expression of stress genes in zebra mussel.

  1. Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, Anna; Faria, Melissa; Barata, Carlos [Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18, 08034 Barcelona (Spain); Pina, Benjamin, E-mail: bpcbmc@cid.csic.e [Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18, 08034 Barcelona (Spain)

    2011-01-15

    Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations. - Coordinated expression of stress genes in zebra mussel.

  2. The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

    Science.gov (United States)

    Heikkila, John J

    2017-01-01

    Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Molecular mechanism of allosteric communication in Hsp70 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Federica Chiappori

    Full Text Available Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD modulate substrate recognition at the Substrate Binding Domain (SBD. Herein, a comparative analysis of an allosteric (Hsp70-DnaK and a non-allosteric structural homolog (Hsp110-Sse1 of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.

  4. Association of plasma IL-6 and Hsp70 with HRV at different levels of PAHs metabolites.

    Directory of Open Access Journals (Sweden)

    Jian Ye

    Full Text Available Exposure to polycyclic aromatic hydrocarbons (PAHs is associated with reduced heart rate variability (HRV, a strong predictor of cardiovascular diseases, but the mechanism is not well understood.We hypothesized that PAHs might induce systemic inflammation and stress response, contributing to altered cardiac autonomic function.HRV indices were measured using a 3-channel digital Holter monitor in 800 coke oven workers. Plasma levels of interleukin-6 (IL-6 and heat shock protein 70 (Hsp70 were determined using ELISA. Twelve urinary PAHs metabolites (OH-PAHs were measured by gas chromatography-mass spectrometry.We found that significant dose-dependent relationships between four urinary OH-PAHs and IL-6 (all Ptrend<0.05; and an increase in quartiles of IL-6 was significantly associated with a decrease in total power (TP and low frequency (LF (Ptrend = 0.014 and 0.006, respectively. In particular, elevated IL-6 was associated in a dose-dependent manner with decreased TP and LF in the high-PAHs metabolites groups (all Ptrend<0.05, but not in the low-PAHs metabolites groups. No significant association between Hsp70 and HRV in total population was found after multivariate adjustment. However, increased Hsp70 was significantly associated with elevated standard deviation of NN intervals (SDNN, TP and LF in the low-PAHs metabolites groups (all Ptrend<0.05. We also observed that both IL-6 and Hsp70 significantly interacted with multiple PAHs metabolites in relation to HRV.In coke oven workers, increased IL-6 was associated with a dose-response decreased HRV in the high-PAHs metabolites groups, whereas increase of Hsp70 can result in significant dose-related increase in HRV in the low-PAHs metabolites groups.

  5. Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

    Science.gov (United States)

    Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

    2017-12-15

    A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

    Science.gov (United States)

    Gentilella, Antonio; Khalili, Kamel

    2011-01-01

    Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

  7. Stress Proteins (hsp70, hsp60) Induced in Isopods and Nematodes by Field Exposure to Metals in a Gradient near Avonmouth, UK

    NARCIS (Netherlands)

    Arts, M.S.J.; Schill, R.O.; Knigge, T.; Eckwert, H.; Kammenga, J.E.; Köhler, H.R.

    2004-01-01

    Heat shock proteins (hsps) are potential biomarkers for monitoring environmental pollution. In this study, the use of hsps as biomarkers in field bioassays was evaluated in terrestrial invertebrates exposed to a metal gradient near Avonmouth, UK. We investigated the hsp70 response in resident and

  8. Species- and Strain-Specific Adaptation of the HSP70 Super Family in Pathogenic Trypanosomatids

    Czech Academy of Sciences Publication Activity Database

    Drini, S.; Criscuolo, A.; Lechat, P.; Imamura, H.; Skalický, Tomáš; Rachidi, N.; Lukeš, Julius; Dujardin, J.-C.; Späth, G.F.

    2016-01-01

    Roč. 8, č. 6 (2016), s. 1980-1995 ISSN 1759-6653 R&D Projects: GA ČR(CZ) GA14-23986S Institutional support: RVO:60077344 Keywords : Leishmania * heat shock protein * HSP70 * evolution * phylogeny * synteny * copy number variation * gene lossgene loss Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.979, year: 2016

  9. The Roles of Heat Shock Proteins 70 and 90 in Exopalaemon carinicauda After WSSV and Vibrio anguillarum Challenges

    Science.gov (United States)

    Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

    2018-04-01

    Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

  10. Hsp60 and p70S6K form a complex in human cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Kroupskaya I. V.

    2011-02-01

    Full Text Available Molecular chaperon Hsp60 and protein kinase p70S6K play an important functional role in the regulation of cardiomyocytes vital function or apoptosis. Aim. To study a possibility of in vivo complex formation between Hsp60 and p70S6K in cardiomyocytes. Methods. Co-immunoprecipitation, Western-blot analysis. Results. We have identified in vivo interaction between molecular chaperone Hsp60 and two isoforms of proteinkinase p70S6K in human myocardium, normal and affected by cardiomyopathy. Conclusions. The results obtained suggest a possible participation of molecular chaperon Hsp60 in regulation of p70S6K activity in stressinduced apoptotic signaling pathway in cardiomyocytes.

  11. Cytosine deletion at AP2-box region of HSP70 promoter and its ...

    Indian Academy of Sciences (India)

    of thermotolerance in cells (Leung et al. 1996). ... region of HSP70 significantly affected cellular thermotol- erance ... The double PCR-RFLP using ScrFI confirmed the occur- ... suade of HSP70 on defense of proteins related to respiration.

  12. Induction of hsp70 by the herbicide oxyfluorfen (Goal) in the Egyptian Nile fish, Oreochromis niloticus.

    Science.gov (United States)

    Hassanein, H M; Banhawy, M A; Soliman, F M; Abdel-Rehim, S A; Müller, W E; Schröder, H C

    1999-07-01

    This paper deals with the expression of the biomarker hsp70 in the liver and kidney of the freshwater fish Oreochromis niloticus following exposure to the herbicide oxyfluorfen (Goal). Fishes were exposed to three concentrations, the 96-h LC50 (3 mg/L), the 96-h (1/2)LC50 (1.5 mg/L), and the 96-h (1/4)LC50 (0.75 mg/L) of oxyfluorfen for 6, 15, and 24 days, respectively, and samples were taken at three different time periods for each concentration. The livers responded to the herbicide by an induction of the expression of both the constitutive (hsp75; Mr 75 kDa) and the inducible (hsp73; Mr 73 kDa) hsp70 proteins. In kidney, the herbicide induced a time-dependent increase in the expression of the constitutive hsp70 (hsp75) as well, but the inducible hsp70 (hsp73) required much longer incubation periods to reach maximal levels (15 and 24 days). Our results suggest that expression of hsp70 in fish is a sensitive indicator of cellular responses to herbicide exposure in the aquatic environment.

  13. Novel polymorphisms in UTR and coding region of inducible heat shock protein 70.1 gene in tropically adapted Indian zebu cattle (Bos indicus) and riverine buffalo (Bubalus bubalis).

    Science.gov (United States)

    Sodhi, M; Mukesh, M; Kishore, A; Mishra, B P; Kataria, R S; Joshi, B K

    2013-09-25

    Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites

  14. Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae.

    Science.gov (United States)

    Germot, A; Philippe, H; Le Guyader, H

    1997-08-01

    In molecular phylogenies based on ribosomal RNA, three amitochondriate protist lineages, Microsporidia, Metamonada (including diplomonads) and Parabasala (including trichomonads), are the earliest offshoots of the eukaryotic tree. As an explantation for the lack of mitochondria in these organisms, the hypothesis that they have diverged before the mitochondrial endosymbiosis is preferred to the less parsimonious hypothesis of several independent losses of the organelle. Nevertheless, if they had descended from mitochondrion-containing ancestors, it may be possible to find in their nuclear DNA genes that derive from the endosymbiont which gave rise to mitochondria. Based on similar evidence, secondary losses of mitochondria have recently been suggested for Entamoeba histolytica and for Trichomonas vaginalis. In this study, we have isolated a gene encoding a chaperone protein (HSP70, 70 kDa heat shock protein) from the microspordian Nosema locustae. In phylogenetic trees, this HSP70 was located within a group of sequences that in other lineages is targetted to the mitochondrial compartment, itself included in the proteobacterial clade. In addition, the N. locustae protein contained the GDAW(V) motif shared by mitochondrial and proteobacterial sequences, with only one conservative substitution. Moreover, microsporidia, a phylum which was assumed to emerge close to the base of the eukaryotic tree, appears as the sister-group of fungi in the HSP70 phylogeny, in agreement with some ultrastructural characters and phylogenies based on alpha- and beta-tubulins. Loss of mitochondria, now demonstrated for several amitochondriate groups, indicates that the common ancestor of all the extant eukaryotic species could have been a mitochondriate eukaryote.

  15. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    International Nuclear Information System (INIS)

    Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

    2007-01-01

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of γH2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si 3 N 4 window showed a homogenous Hsp70 expression pattern

  16. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

    2007-07-15

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

  17. [Association of occupational chronic psychological stress with heat shock protein 70 in serum and tumor necrosis factor-alpha expression levels].

    Science.gov (United States)

    Qiu, F Y; Tian, R L; Qiang, Y; He, K P; Liu, H R; Zhang, W; Song, H

    2016-05-01

    To investigate the relationship between occupational chronic psychological stress with heat shock protein 70 (HSP70) and tumor necrosis factor-alpha (TNF-α). Using case-control study design, we selected 622 cases in 20 to 60 years old and unrelated patients with metabolic syndrome as the case group between October 2011 and October 2012 at two hospitals of Ningxia hui autonomous region. At the same time, we selected 600 healthy people from health check-up crowd in the above two hospitals as control group. The the research objects were sex, age, nation, height, weight, smoking, drinking, exercise, and so on. After informed consent, all the research objects were collected fasting venous blood samples 10 ml in order to proceed laboratory testing of biochemical indicators. The expression of HSP70 and TNF-α in serum was determined by ELISA. Using the revised occupational stress inventory (OSI) to survey the occupational chronic psychological stress factors and stress level of research object. The correlation of occupational chronic psychological stress scores with HSP70 and TNF-α was investigated by partial correlation analysis. We built a multivariate linear regression equation With HSP70 and TNF alpha as the independent variable and occupational chronic psychological stress scores as the dependent variable, using equation of the determination coefficient R(2) to judge the degree of fitting equation. The total points of chronic stress factors in all respondents was (136.65±16.19). Among them, the mild stress level group was 313, moderate was 588, severe was 321, chronic heart stress factors scores were (119.96±13.30), (135.33±3.23), (155.33±13.55) points, respectively. In the case group subjects, the expression of HSP70 in mild, moderate and severe occupational chronic psychological stress levels were (29.88±30.08), (36.38±30.08), (27.16±23.77) ng/ml (F=6.85, P=0.001). The control group were (27.64±9.89), (39.78±29.77), (3.94±3.09) ng/ml (F=125.71, Pstress

  18. Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats

    Directory of Open Access Journals (Sweden)

    Shevtsov MA

    2014-05-01

    Full Text Available Maxim A Shevtsov,1,2 Boris P Nikolaev,3 Ludmila Y Yakovleva,3 Anatolii V Dobrodumov,4 Anastasiy S Dayneko,5 Alexey A Shmonin,5,6 Timur D Vlasov,5 Elena V Melnikova,5 Alexander D Vilisov,4,5 Irina V Guzhova,1 Alexander M Ischenko,3 Anastasiya L Mikhrina,7 Oleg V Galibin,5 Igor V Yakovenko,2 Boris A Margulis1 1Institute of Cytology of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 2AL Polenov Russian Research Scientific Institute of Neurosurgery, St Petersburg, Russia; 3Research Institute of Highly Pure Biopreparations, St Petersburg, Russia; 4Institute of Macromolecular Compounds of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 5First St Petersburg IP Pavlov State Medical University, St Petersburg, Russia; 6Federal Almazov Medical Research Centre, St Petersburg, Russia; 7IM Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (RAS, St Petersburg, Russia Abstract: Recombinant 70 kDa heat shock protein (Hsp70 is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg. To assess Hsp70’s neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia. Rats were then kept alive for 72 hours. The

  19. IL-10 is significantly involved in HSP70-regulation of experimental subretinal fibrosis.

    Directory of Open Access Journals (Sweden)

    Yang Yang

    Full Text Available Subretinal fibrosis is directly related to severe visual loss, especially if occurs in the macula, and is frequently observed in advanced age-related macular degeneration and other refractory eye disorders such as diabetic retinopathy and uveitis. In this study, we analyzed the immunosuppressive mechanism of subretinal fibrosis using the novel animal model recently demonstrated. Both TLR2 and TLR4 deficient mice showed significant enlargement of subretinal fibrotic area as compared with wild-type mice. A single intraocular administration of heat shock protein 70 (HSP70, which is an endogenous ligand for TLR2 and TLR4, inhibited subretinal fibrosis in wild-type mice but not in TLR2 and TLR4-deficient mice. Additionally, HSP70 induced IL-10 production in eyes from wild-type mice but was impaired in both TLR2- and TLR4-deficient mice, indicating that HSP70-TLR2/TLR4 axis plays an immunomodulatory role in subretinal fibrosis. Thus, these results suggest that HSP70-TLR2/TLR4 axis is a new therapeutic target for subretinal fibrosis due to prognostic CNV.

  20. Calpain 3 and CaMKIIβ signaling are required to induce HSP70 necessary for adaptive muscle growth after atrophy

    Science.gov (United States)

    Kramerova, Irina; Torres, Jorge A; Eskin, Ascia; Nelson, Stanley F; Spencer, Melissa J

    2018-01-01

    Abstract Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIβ signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIβ signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIβ regulates remodeling in multiple contexts: endurance exercise and growth after atrophy. PMID:29528394

  1. The role of heat shock protein 70 in oxidant stress and inflammatory injury in quail spleen induced by cold stress.

    Science.gov (United States)

    Ren, Jiayi; Liu, Chunpeng; Zhao, Dan; Fu, Jing

    2018-05-15

    The aim of this study was to investigate the role of heat shock protein 70 (Hsp70) in oxidative stress and inflammatory damage in the spleen of quails which were induced by cold stress. One hundred ninety-two 15-day-old male quails were randomly divided into 12 groups and kept at 12 ± 1 °C to examine acute and chronic cold stress. We first detected the changes in activities of antioxidant enzymes in the spleen tissue under acute and chronic cold stress. The activities of glutathione peroxidase (GSH-Px) fluctuated in acute cold stress groups, while they were significantly decreased (p stress. The activities of superoxide dismutase (SOD), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) content were decreased significantly (p stress groups. Malondialdehyde (MDA) content was significantly increased (p stress except the 0.5 h group of acute cold stress. Besides, histopathological analysis showed that quail's spleen tissue was inflammatory injured seriously in both the acute and chronic cold stress groups. Additionally, the inflammatory factors (cyclooxygenase-2 (COX-2), prostaglandin E synthase (PTGES), iNOS, nuclear factor-kappa B (NF-κB), and tumor necrosis factor-a (TNF-α)) and Hsp70 mRNA levels were increased in both of the acute and chronic cold stress groups compared with the control groups. These results suggest that oxidative stress and inflammatory injury could be induced by cold stress in spleen tissues of quails. Furthermore, the increased expression of Hsp70 may play a role in protecting the spleen against oxidative stress and inflammatory damage caused by cold stress.

  2. A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors.

    OpenAIRE

    Craven, R A; Egerton, M; Stirling, C J

    1996-01-01

    The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones. We report that this 881 codon reading frame represents a functional gene expressing a 113-119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER). We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy). Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced...

  3. Cell type-specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia.

    Science.gov (United States)

    Oehler, R; Pusch, E; Zellner, M; Dungel, P; Hergovics, N; Homoncik, M; Eliasen, M M; Brabec, M; Roth, E

    2001-10-01

    Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

  4. Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

    International Nuclear Information System (INIS)

    Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

    2011-01-01

    Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT TM ). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000 μM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.

  5. The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

    2008-09-19

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

  6. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology.

    Science.gov (United States)

    Kiang, J G; Tsokos, G C

    1998-11-01

    Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic. In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults. Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer. Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases. Most of the HSPs interact with other proteins in cells and alter their function. These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions. In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways. The clinical importance and therapeutic potential of HSPs are discussed.

  7. The expression of heat shock proteins 70 and 90 in pea seedlings under simulated microgravity conditions

    Science.gov (United States)

    Kozeko, L.

    Microgravity is an abnormal and so stress factor for plants. Expression of known stress-related genes is appeared to implicate in the cell response to different kinds of stress. Heat shock proteins HSP70 and HSP90 are present in plant cells under the normal growth conditions and their quantity increases during stress. The effect of simulated microgravity on expression of HSP70 and HSP90 was studied in etiolated Pisum sativum seedlings grown on the horizontal clinostat (2 rpm) from seed germination for 3 days. Seedlings were also subjected to two other types of stressors: vertical clinorotatoin (2 rpm) and 2 h temperature elevation (40°C). HSPs' level was measured by ELISA. The quantity of both HSPs increased more than in three times in the seedlings on the horizontal clinostat in comparison with the stationary 1 g control. Vertical clinorotation also increased HSPs' level but less at about 20% than horizontal one. These effects were comparable with the influence of temperature elevation. The data presented suggest that simulated microgravity upregulate HSP70 and HSP90 expression. The increased HSPs' level might evidence the important functional role of these proteins in plant adaptation to microgravity. We are currently investigating the contribution of constitutive or inducible forms of the HSPs in this stress response.

  8. Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

    Science.gov (United States)

    Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A

    2015-04-10

    Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response.

    Science.gov (United States)

    Zhang, Hong; Yang, Jie; Wu, Si; Gong, Weibin; Chen, Chang; Perrett, Sarah

    2016-03-25

    DnaK is the major bacterial Hsp70, participating in DNA replication, protein folding, and the stress response. DnaK cooperates with the Hsp40 co-chaperone DnaJ and the nucleotide exchange factor GrpE. Under non-stress conditions, DnaK binds to the heat shock transcription factor σ(32)and facilitates its degradation. Oxidative stress results in temporary inactivation of DnaK due to depletion of cellular ATP and thiol modifications such as glutathionylation until normal cellular ATP levels and a reducing environment are restored. However, the biological significance of DnaK glutathionylation remains unknown, and the mechanisms by which glutathionylation may regulate the activity of DnaK are also unclear. We investigated the conditions under which Escherichia coli DnaK undergoesS-glutathionylation. We observed glutathionylation of DnaK in lysates of E. coli cells that had been subjected to oxidative stress. We also obtained homogeneously glutathionylated DnaK using purified DnaK in the apo state. We found that glutathionylation of DnaK reversibly changes the secondary structure and tertiary conformation, leading to reduced nucleotide and peptide binding ability. The chaperone activity of DnaK was reversibly down-regulated by glutathionylation, accompanying the structural changes. We found that interaction of DnaK with DnaJ, GrpE, or σ(32)becomes weaker when DnaK is glutathionylated, and the interaction is restored upon deglutathionylation. This study confirms that glutathionylation down-regulates the functions of DnaK under oxidizing conditions, and this down-regulation may facilitate release of σ(32)from its interaction with DnaK, thus triggering the heat shock response. Such a mechanism provides a link between oxidative stress and the heat shock response in bacteria. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

    2008-01-01

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

  11. Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

    Science.gov (United States)

    Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C

    2009-04-15

    Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

  12. NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

    Science.gov (United States)

    Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

    2018-01-01

    Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

  13. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites

    Science.gov (United States)

    Charnaud, Sarah C.; Dixon, Matthew W. A.; Nie, Catherine Q.; Chappell, Lia; Sanders, Paul R.; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J.; Beeson, James G.; Rayner, Julian C.; Przyborski, Jude M.; Tilley, Leann; Crabb, Brendan S.

    2017-01-01

    Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins. PMID:28732045

  14. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

    Directory of Open Access Journals (Sweden)

    Sarah C Charnaud

    Full Text Available Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

  15. The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein.

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    Daniel W Summers

    Full Text Available Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP. The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

  16. The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR Analysis

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    Daniela Fiocco

    2011-08-01

    Full Text Available Small heat shock proteins (sHsps are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR procedure was developed and used to quantify the transcript level of a small heat shock gene (shs in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C, bile (0.3% w/v, hyperosmosis (1 M and 2.5 M NaCl, and low pH value (pH 4. The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR sequence (TTAGCACTC-N9-GAGTGCTAA homologue to the controlling IR of chaperone expression (CIRCE elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

  17. DnaK as Antibiotic Target: Hot Spot Residues Analysis for Differential Inhibition of the Bacterial Protein in Comparison with the Human HSP70.

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    Federica Chiappori

    Full Text Available DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50-70%, which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.

  18. Cytotoxicity and Hsp 70 induction in Hep G2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock

    International Nuclear Information System (INIS)

    Hassen, Wafa; Golli, Emna El; Baudrimont, Isabelle; Mobio, A. Theophile; Ladjimi, M. Moncef; Creppy, E. Edmond; Bacha, Hassen

    2005-01-01

    Zearalenone (ZEN) is a mycotoxin with several adverse effects in laboratory and domestic animals. The mechanism of ZEN toxicity that involves mainly binding to oestrogen receptors and inhibition of macromolecules synthesis is not fully understood. Using human hepatocytes Hep G2 cells as a model, the aim of this work was (i) to investigate the ability of ZEN to induce heat shock proteins Hsp 70 and (ii) to find out the mechanisms of ZEN cytotoxicity by examining cell proliferation and protein synthesis. Our study demonstrated that ZEN induces Hsp 70 expression in a time and dose-dependant manner; this induction occurs at non-cytotoxic concentrations, it could be therefore considered as a biomarker of toxicity. A cytoprotective effect of Hsp 70 was elicited when Hep G2 cells were exposed to Sub-Lethal heat shock prior to ZEN treatment and evidenced by a reduced ZEN cytolethality. This cytoprotection suggests that Hsp 70 may constitute an important cellular defence mechanism. Finally, our data show that ZEN is cytotoxic in Hep G2 cells by inhibiting cell proliferation and total protein synthesis and pointed out oxidative damage as possible pathway involved in ZEN toxicity; however, other investigations are needed to further confirm Zen induced oxidative stress

  19. Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

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    J Dhakal

    2013-01-01

    Full Text Available Background: Heat shock proteins (Hsps are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. Objectives: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70 as adjuvant or antigen carrier. Materials and Methods: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70 amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. Results: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI. Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund′s adjuvant. Conclusion: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund′s adjuvant. Further, studies need to be carried out to explore its

  20. Influence of Moringa oleifera extract, vitamin C, and sodium bicarbonate on heat stress-induced HSP70 expression and cellular immune response in rabbits.

    Science.gov (United States)

    Abdel-Latif, Mahmoud; Sakran, Thabet; Badawi, Yasser K; Abdel-Hady, Doaa S

    2018-05-04

    The current study aimed to test the effect of Moringa oleifera extract (MOE), vitamin (Vit) C, and sodium bicarbonate (NaHCO 3 ) on heat stress (HS)-induced alterations in rabbits. Five groups of rabbits were designed as control, HS, HS + MOE, HS + Vit C, and HS + NaHCO 3 . HS groups were exposed to high temperatures, while treatments were given in drinking water for 6 weeks. Levels of blood cortisol, leptin, IFN-γ, TNF-α, and IL-10 were assayed using ELISA, while adrenaline was assayed calorimetrically. Expression of HSP70, FOXP3, T cell receptor (TCR) γ, and δ mRNA was tested using real-time (RT)-PCR, while HSP70 protein expression was tested using western blotting in liver and kidney tissues. Infiltration of regulatory T cells (Treg; CD25 + ) and NK (CD56 + ) cells were tested using immunohistochemistry (IHC). The levels of liver enzymes (ALT & AST), urea, and creatinine were assayed calorimetrically, while body weight gain (BWG) and feed conversion ratio (FCR) were calculated. The results showed increased levels of cortisol, adrenaline, leptin, IFN-γ, TNF-α, ALT, AST, urea, and creatinine but decreased IL-10 in the HS group. Increased expression of HSP70 on both mRNA and protein levels was associated with increased NK and γδ T cells versus decreased Treg cell infiltration in liver and kidney tissues of the HS group. In the same group, BWG was decreased, while FCR was increased with respect to the control group. All treatments used in this study reversed the effects of HS significantly. In conclusion, MOE, Vit C, and NaHCO 3 can be added to rabbit diets for the amelioration of HS-induced symptoms.

  1. Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

    Science.gov (United States)

    Montalvo Alvarez, Ana Margarita; Nodarse, Jorge Fraga; Goodridge, Ivón Montano; Fidalgo, Lianet Monzote; Marin, Marcel; Van Der Auwera, Gert; Dujardin, Jean-Claude; Bernal, Iván Darío Velez; Muskus, Carlos

    2010-05-01

    Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

  2. Expression of heat shock protein 70 in transport-stressed broiler pectoralis major muscle and its relationship with meat quality.

    Science.gov (United States)

    Xing, T; Wang, M F; Han, M Y; Zhu, X S; Xu, X L; Zhou, G H

    2017-09-01

    Omics research has indicated that heat shock protein 70 (HSP70) is a potential biomarker of meat quality. However, the specific changes and the potential role of HSP70 in postmortem meat quality development need to be further defined. In this study, Arbor Acres broiler chickens (n=126) were randomly categorized into three treatment groups of unstressed control (C), 0.5-h transport (T) and subsequent water shower spray following transport (T/W). Each treatment consisted of six replicates with seven birds each. The birds were transported according to a designed protocol. The pectoralis major (PM) muscles of the transport-stressed broilers were categorized as normal and pale, soft and exudative (PSE)-like muscle samples according to L* and pH24 h values to test the expression and location of HSP70. Results revealed that the activities of plasma creatine kinase and lactate dehydrogenase increased significantly (Pmeat quality and stress indicators. In conclusion, this research suggests that the variation in HSP70 expression may provide a novel insight into the pathways underlying meat quality development.

  3. Complex patterns of geographic variation in heat tolerance and Hsp70 expression levels in the common frog Rana temporaria

    DEFF Research Database (Denmark)

    Sørensen, Jesper Givskov; Pekkonen, Minna; Lindgren, Beatrice

    2009-01-01

    1. We tested for geographical variation in heat tolerance and Hsp70 expression levels of Rana temporaria tadpoles along a 1500 km long latitudinal gradient in Sweden.   2. Temperature tolerance of the hatchling tadpoles did not differ among populations, but they tolerated stressful hot temperatur...

  4. Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

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    Trevor M. Morey

    2017-12-01

    Full Text Available Choline acetyltransferase (ChAT synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS. One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A or mutation of residue Val18 (V18M enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID, co-immunoprecipitation and in situ proximity-ligation assay (PLA, we identified the heat shock proteins (HSPs HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms

  5. Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Mingle; Zou, Zhongwei; Li, Qinghui; Xin, Huahong; Zhu, Xujun; Chen, Xuan; Li, Xinghui

    2017-07-01

    CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

  6. Ekspresi Human Leukocyte Antigen-G (HLA-G dan Heat-Shock Protein-70 (Hsp-70 pada Pertumbuhan Janin Terhambat

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    Sri Sulistyowati

    2014-03-01

    Full Text Available Intra uterine growth retardation (IUGR is one of the leading causes of higher morbidity and mortality in perinatal. Immune maladaptation affects trophoblast invasion and spiralis arteria remodeling that will cause placental tissue hypoxia. This research aimed to analyze human leukocyte antigen-G (HLA-G and heat-shock protein-70 (Hsp-70 expression on the IUGR trophoblast and normal pregnancy, by applying analytical observational method and cross sectional approach. This research was conducted at the Obstetric and Gynecology Department of Dr. Moewardi Hospital Surakarta from November to December 2011. The total samples were 30, divided into two groups. There were 15 samples trophoblast on IUGR and 15 samples trophoblast from normal pregnancy. All samples were tested for HLA-G and Hsp-70 using immunohistochemistry. The data were analyzed by using t-test. The mean of HLA-G expression on the IUGR groups was 32.42±8.90 and on the normal pregnancy groups was 43.92±14.91 (p=0.016. Heat-shock protein70 expression on the IUGR groups was 2.4355+0.26647 and on the normal pregnancy groups was 1.5920+0.17142 with p=0.008. In conclusion, in IUGR, the HLA-G expression is lower and the Hsp-70 expression is higher than in normal pregnancy.

  7. AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA-dependent and independent signalling to attenuate plant response to abiotic stress.

    Science.gov (United States)

    Sun, Xinbo; Sun, Chunyu; Li, Zhigang; Hu, Qian; Han, Liebao; Luo, Hong

    2016-06-01

    Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post-germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis-related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA-independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA-dependent and independent signalling pathways. © 2015 John Wiley & Sons Ltd.

  8. Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

    Science.gov (United States)

    Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

    1997-03-15

    The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

  9. A Recombinant Trivalent Fusion Protein F1-LcrV-HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis.

    Science.gov (United States)

    Verma, Shailendra K; Batra, Lalit; Tuteja, Urmil

    2016-01-01

    Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

  10. Characterization of novel heat-responsive transcription factor (TaHSFA6e) gene involved in regulation of heat shock proteins (HSPs) - A key member of heat stress-tolerance network of wheat.

    Science.gov (United States)

    Kumar, Ranjeet R; Goswami, Suneha; Singh, Khushboo; Dubey, Kavita; Rai, Gyanendra K; Singh, Bhupinder; Singh, Shivdhar; Grover, Monendra; Mishra, Dwijesh; Kumar, Sanjeev; Bakshi, Suman; Rai, Anil; Pathak, Himanshu; Chinnusamy, Viswanathan; Praveen, Shelly

    2018-08-10

    Heat stress has an adverse effect on the quality and quantity of agriculturally important crops, especially wheat. The tolerance mechanism has not been explored much in wheat and very few genes/ TFs responsive to heat stress is available on public domain. Here, we identified, cloned and characterized a putative TaHSFA6e TF gene of 1.3 kb from wheat cv. HD2985. We observed an ORF of 368 aa with Hsf DNA binding signature domain in the amino acid sequence. Single copy number of TaHSFA6e was observed integrated in the genome of wheat. Expression analysis of TaHSFA6e under differential HS showed maximum transcripts in wheat cv. Halna (thermotolerant) in response to 38 °C for 2 h during pollination and grain-filling stages, as compared to PBW343, HD2329 and HD2985. Putative target genes of TaHSFA6e (HSP17, HSP70 and HSP90) showed upregulation in response to differential HS (30 & 38 °C, 2 h) during pollination and grain-filling stages. Small HSP17 was observed most triggered in Halna under HS. We observed increase in the catalase, guaiacol peroxidase, total antioxidant capacity (TAC), and decrease in the lipid peroxidation in thermotolerant cvs. (Halna, HD2985), as compared to thermosusceptible (PBW343, HD2329) under differential HS. Multiple stresses (heat - 38 °C, 2 h, and drought - 100 mL of 20% polyethylene Glycol 6000) during seedling stage of wheat showed positive correlation between the expression of TaHSFA6e, putative targets (HSP70, HSP90, HSP17) and TAC. Halna (thermotolerant) performed better, as compared to other contrasting cvs. TaHSFA6e TF can be used as promising candidate gene for manipulating the heat stress-tolerance network. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Hsp27, Hsp70 and mismatch repair proteins hMLH1 and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients.

    Science.gov (United States)

    Nadin, Silvina Beatriz; Vargas-Roig, Laura M; Drago, Gisela; Ibarra, Jorge; Ciocca, Daniel R

    2007-07-08

    Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival

  12. Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J

    2015-09-01

    Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

    Science.gov (United States)

    Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

    2018-04-01

    Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

  14. Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Sarah M Senf

    Full Text Available Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70 is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our

  15. Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast.

    Science.gov (United States)

    Gao, Caiqiu; Jiang, Bo; Wang, Yucheng; Liu, Guifeng; Yang, Chuanping

    2012-04-01

    It is well known that plant heat shock proteins (HSPs) play important roles both in response to adverse environmental conditions and in various developmental processes. However, among plant HSPs, the functions of tree plant HSPs are poorly characterized. To improve our understanding of tree HSPs, we cloned and characterized an HSP gene (ThHSP18.3) from Tamarix hispida. Sequence alignment reveals that ThHSP18.3 belongs to the class I small heat shock protein family. A transient expression assay showed that ThHSP18.3 protein was targeted to the cell nucleus. Treatment of Tamarix hispida with cold and heat shock highly induced ThHSP18.3 expression in all studied leaves, roots and stems, whereas, treatment of T. hispida with NaCl, NaHCO(3), and PEG induced ThHSP18.3 expression in leaves and decreased its expression in roots and stems. Further, to study the role of ThHSP18.3 in stress tolerance under different stress conditions, we cloned ThHSP18.3 into the pYES2 vector, transformed and expressed the vector in yeast Saccharomyces cerevisiae. Yeast cells transformed with an empty pYES2 vector were employed as a control. Compared to the control, yeast cells expressing ThHSP18.3 showed greater tolerance to salt, drought, heavy metals, and both low and high temperatures, indicating that ThHSP18.3 confers tolerance to these stress conditions. These results suggested that ThHSP18.3 is involved in tolerance to a variety of stress conditions in T. hispida.

  16. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

    Science.gov (United States)

    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  17. Heat-Induced Changes in Heat Shock Protein Genes Expression in Crossbred and Baladi Pregnant Cows and Their Offspring

    International Nuclear Information System (INIS)

    Khalil, W.K.B.; Nessim, M.Z.; El- Masry, K.A.

    2010-01-01

    The experiment was carried out during August (hot climate) on twelve pregnant cows of six crossbred (50% native Baladi and 50% Brown Swiss) and six native Baladi pregnant cows in the same age and the second parity during their mid-pregnancy as detected by rectal palpation. The experiment was repeated during December (mild climate) on similar twelve pregnant cows. Blood sample was obtained from each cow at the end of August (first group) and at the end of December (second group) to obtain heat shock protein genes expression (HSP72, HSP70.01, HSP70, HSP47, k Dalton and Actin) in pregnant cows under mild and hot climate to find out, which breed is more tolerant to heat stress and to estimate offspring birth weight and their growth performances during suckling period. Comparison was made between hot climate cows group and mild climate cows group to estimate heat- induced changes in both breeds in expression level of the Hsp genes and to compare with their neonate birth weight and growth performances during suckling period. The results revealed that expression level of the Hsp genes (Hsp72, Hsp70.1, Hsp70 and Hsp 47) was higher (p<0.01) in hot season compared to that of mild season. Expression level of the Hsp genes (Hsp70.1, Hsp70 and Hsp 47) was higher (p<0.05) in crossbred cows than in Baldi cows under summer hot season. This indicates that crossbred cows are less heat tolerant than Baladi cows under heat stress climate. Heat induced decrease (p<0.01) in offspring birth weight in Baladi and crossbred by 18.1% and 25%, respectively, in weaning weight by 14.61% and 23.14%, respectively and in body weight gain by 14.61% and 21.18%, respectively

  18. Mercury induced oxidative stress, DNA damage, and activation of antioxidative system and Hsp70 induction in duckweed (Lemna minor).

    Science.gov (United States)

    Zhang, Tingting; Lu, Qianqian; Su, Chunlei; Yang, Yaru; Hu, Dan; Xu, Qinsong

    2017-09-01

    Mercury uptake and its effects on physiology, biochemistry and genomic stability were investigated in Lemna minor after 2 and 6d of exposure to 0-30μM Hg. The accumulation of Hg increased in a concentration- and duration-dependent manner, and was positively correlated with the leaf damage. Oxidative stress after Hg exposure was evidenced in L. minor by a significant decrease in photosynthetic pigments, an increase in malondialdehyde and lipoxygenase activities (total enzyme activity and isoenzymes activity). Fronds of L. minor exposed to Hg showed an induction of peroxidase, catalase, and ascorbate peroxidase activities (total enzyme activity and some isoenzymes activities). Exposure of L. minor to Hg reduced the activity (total enzyme activity and some isoenzymes activities) of glutathione reductase, and superoxide dismutase. Exposure to Hg produced a transient increase in the content of glutathione and ascorbic acid. The content of dehydroascorbate and oxidized glutathione in L. minor were high during the entire exposure period. Exposure of L. minor to Hg also caused the accumulation of proline and soluble sugars. The amplification of new bands and the absence of normal DNA amplicons in treated plants in the random amplified polymorphic DNA (RAPD) profile indicated that genomic template stability (GTS) was affected by Hg treatment. The accumulation of Hsp70 indicated the occurrence of a heat shock response at all Hg concentrations. These results suggest that L. minor plants were able to cope with Hg toxicity through the activation of various mechanisms involving enzymatic and non-enzymatic antioxidants, up-regulation of proline, and induction of Hsp70. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The association of SNPs in Hsp90β gene 5' flanking region with thermo tolerance traits and tissue mRNA expression in two chicken breeds.

    Science.gov (United States)

    Chen, Zhuo-Yu; Gan, Jian-Kang; Xiao, Xiong; Jiang, Li-Yan; Zhang, Xi-Quan; Luo, Qing-Bin

    2013-09-01

    Thermo stress induces heat shock proteins (HSPs) expression and HSP90 family is one of them that has been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the Hsp90β gene in chicken with thermo tolerance is still unknown. Direct sequencing was used to detect possible SNPs in Hsp90β gene 5' flanking region in 3 chicken breeds (n = 663). Six mutations, among which 2 SNPs were chosen and genotypes were analyzed with PCR-RFLP method, were found in Hsp90β gene in these 3 chicken breeds. Association analysis indicated that SNP of C.-141G>A in the 5' flanking region of the Hsp90β gene in chicken had some effect on thermo tolerance traits, which may be a potential molecular marker of thermo tolerance, and the genotype GG was the thermo tolerance genotype. Hsp90β gene mRNA expression in different tissues detected by quantitative real-time PCR assay were demonstrated to be tissue dependent, implying that different tissues have distinct sensibilities to thermo stress. Besides, it was shown time specific and varieties differences. The expression of Hsp90β mRNA in Lingshan chickens in some tissues including heart, liver, brain and spleen were significantly higher or lower than that of White Recessive Rock (WRR). In this study, we presume that these mutations could be used in marker assisted selection for anti-heat stress chickens in our breeding program, and WRR were vulnerable to tropical thermo stress whereas Lingshan chickens were well adapted.

  20. Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

    Directory of Open Access Journals (Sweden)

    Michael Reidy

    2014-10-01

    Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

  1. Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

    Science.gov (United States)

    Yu, Hong; Bao, En-Dong; Zhao, Ru-Qian; Lv, Qiong-Xia

    2007-11-01

    To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. 24 pigs (mean weight, 20 +/- 1 kg). Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

  2. Inhibition of inducible heat shock protein-70 (hsp72 enhances bortezomib-induced cell death in human bladder cancer cells.

    Directory of Open Access Journals (Sweden)

    Wei Qi

    Full Text Available The proteasome inhibitor bortezomib (Velcade is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1A(high cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1A(low cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1 to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1A(low cell lines (UM-UC10 and UM-UC13, and exposure to the chromatin demethylating agent 5-aza-2'-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B-V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.

  3. Geranylgeranylacetone ameliorates lung ischemia/reperfusion injury by HSP70 and thioredoxin redox system: NF-kB pathway involved.

    Science.gov (United States)

    Cao, Weijun; Li, Manhui; Li, Jianxiong; Li, Chengwei; Xu, Xin; Gu, Weiqing

    2015-06-01

    Geranylgeranylacetone (GGA) has been clinically used as an anti-ulcer drug. In the present study, we explored the protective effects of GGA on lung ischemia/reperfusion injury (IRI) and the underlying mechanism. The results demonstrated that GGA ameliorated the lung biochemical and histological alterations induced by IRI, which was reversed by HSP70 inhibition. To further explore the mechanism of GGA action, we focused on NF-kB and thioredoxin (Trx) redox system. It was shown that GGA induced the HSP70 and Trx-1 expression, NF-kB nuclear translocation and activated thioredoxin reductase (TrxR). The Trx-1 expression and TrxR activity was suppressed by HSP70 and NF-kB inhibition, while the nuclear NF-kB p65 expression was suppressed by HSP70 inhibitor. These results indicated that GGA may protect rat lung against IRI by HSP70 and Trx redox system, in which NF-kB pathway may be involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

    Science.gov (United States)

    Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

    2013-05-29

    The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

  5. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Energy Technology Data Exchange (ETDEWEB)

    Zorzi, Elisa [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Bonvini, Paolo, E-mail: paolo.bonvini@unipd.it [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Fondazione Città della Speranza, 36030 Monte di Malo, Vicenza (Italy)

    2011-10-21

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

  6. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Science.gov (United States)

    Zorzi, Elisa; Bonvini, Paolo

    2011-01-01

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells. PMID:24213118

  7. A comparison of piezosurgery and conventional surgery by heat shock protein 70 expression.

    Science.gov (United States)

    Gülnahar, Y; Hüseyin Köşger, H; Tutar, Y

    2013-04-01

    The effects of mechanical instruments on the viability of cells are essential in terms of regeneration. There is considerable interest in cell repair following damage mediated by dental surgical procedures. Cells can tolerate stress by expressing heat shock protein 70 (Hsp70). During and after surgical tooth removal, oxidative stress can activate Hsp70 expression proportional to the intensity of the stress signal stimulus to cope with stress. This study examined the expression of Hsp70 as a potential biomarker of immediate postoperative stress in patients undergoing two different surgical procedures of different severity. Expression of Hsp70 both at mRNA and at protein level in the conventional group was two-fold higher than that of the piezo group. This suggests that tooth movement by the piezo method causes relatively lower stress in the alveolar bone. Piezosurgery provides relatively low stress to the patients and this may help cell repair after the surgical procedure. Patients undergoing more aggressive surgery using conventional methods showed a significant increase in Hsp70 in the immediate postoperative period. Therefore, Hsp70 induction can be a potential tool as a prognostic surgical marker. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  8. Adult Heat Tolerance Variation in Drosophila melanogaster is Not Related to Hsp70 Expression

    DEFF Research Database (Denmark)

    Jensen, Louise Toft; Cockerell, Fiona Elizabeth; Kristensen, Torsten Nygaard

    2010-01-01

    in Drosophila larvae Hsp70 expression may be a key determinant of heat tolerance, the evidence for this in adults is equivocal. We therefore examined heat-induced Hsp70 expression and several measurements of adult heat tolerance in three independent collections of D. melanogaster, measured in three laboratories...

  9. Amelioration of Heat-Stress Conditions of Egyptian Summer Season on Friesian Calves Using Air Condition

    International Nuclear Information System (INIS)

    Nessim, M.Z.; Kamal, T.H.; Khalil, W.K.B.

    2010-01-01

    Male Friesian calves were used to evaluate cool air condition (AC) in alleviating heat stress (HS) determined by Heat Shock Protein genes expression (HSP), hormonal, biochemical and physiological parameters. The animals were exposed to summer heat stress (HS) under shade for two weeks (control). The maximum temperature humidity index (THI) during summer HS was from 81 to 88. Afterward the animals were exposed to AC, inside a climatic chamber for 6 hours daily for two weeks, where, the THI was from 70 to 71. The results revealed that expression level of the Hsp genes (Hsp72, Hsp70.1, Hsp70 and Hsp47) was lower under air condition treatment than under summer heat stress. Rectal temperature and respiration rate were significantly lower (p< 0.01) under air condition treatment than those under heat stress. Total triiodothyronin (T3) level was significantly higher (P< 0.05) in AC cooling treatments than in HS, while cortisol level was significantly lower (P < 0.01) in AC cooling treatment than in HS calves. Creatinine and Urea -N levels were significantly lower (P < 0.01) in AC cooling treatment than in HS calves. Triglycerides, ALT and AST levels were significantly lower (p<0.01), (P< 0.01) and (p<0.05), respectively in AC cooling treatment than in HS calves. These results demonstrated that there is a relationship between the molecular weight of HSPs and the level of HSPs gene exprisson. The higher the molecular weight (HSP 72) the lower is the HSPs gene expression level (0.82 in HS and 0.39 in AC) and vise versa. This holds true in both heat stress and air condition. AC treatment is capable to ameliorate heat stress of Friesian calves under hot summer climate

  10. Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy

    Science.gov (United States)

    Jheng, Huei-Fen; Tsai, Pei-Jane; Chuang, Yi-Lun; Shen, Yi-Ting; Tai, Ting-An; Chen, Wen-Chung; Chou, Chuan-Kai; Ho, Li-Chun; Tang, Ming-Jer; Lai, Kuei-Tai A.; Sung, Junne-Ming; Tsai, Yau-Sheng

    2015-01-01

    ABSTRACT Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4−/− mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN. PMID:26398934

  11. The Hsp60C gene in the 25F cytogenetic region in Drosophila ...

    Indian Academy of Sciences (India)

    Unknown

    Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the ..... C. Genomic organization and the predicted.

  12. Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus

    Directory of Open Access Journals (Sweden)

    Schoville Sean D

    2012-09-01

    Full Text Available Abstract Background Geographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance. Results Observed differences in gene expression between the southern (San Diego and the northern (Santa Cruz populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps and genes involved in ubiquitination and proteolysis. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Among other sets of genes, cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were down-regulated in both populations. Conclusions Marked changes in gene expression were observed in response to acute sub-lethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations, the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s involved in acute temperature stress may offer at

  13. Effect of Hypergravity on the Level of Heat Shock Proteins 70 and 90 in Pea Seedlings

    Science.gov (United States)

    Kozeko, Liudmyla; Kordyum, Elizabeth

    2009-01-01

    Exposure to hypergravity induces significant changes in gene expression of plants which are indicative of stress conditions. A substantial part of the general stress response is up-regulation of heat shock proteins (Hsp) which function as molecular chaperones. The objective of this research was to test the possible changes in the Hsp70 and Hsp90 level in response to short-term hypergravity exposure. In this study 5-day-old etiolated pea seedlings were exposed to centrifuge-induced hypergravity (3-14 g) for 15 min and 1 h and a part of the seedlings was sampled at 1.5 and 24 h after the exposures. Western blot analysis showed time-dependent changes in Hsp70 and Hsp90 levels: an increase under hypergravity and a tendency towards recovery of the normal content during re-adaptation. The quantity and time of their expression was correlated with the g-force level. These data suggest that short-term hypergravity acts as a stress which could increase the risk of protein denaturation and aggregation. Molecular chaperons induced during the stress may have an essential role in counteracting this risk.

  14. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

    Directory of Open Access Journals (Sweden)

    Fresno Manuel

    2011-07-01

    Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

  15. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...

  16. Radioresistance of human glioma spheroids and expression of HSP70, p53 and EGFr

    International Nuclear Information System (INIS)

    Fedrigo, Carlos A; Rocha, Adriana B da; Grivicich, Ivana; Schunemann, Daniel P; Chemale, Ivan M; Santos, Daiane dos; Jacovas, Thais; Boschetti, Patryck S; Jotz, Geraldo P; Filho, Aroldo Braga

    2011-01-01

    Radiation therapy is routinely prescribed for high-grade malignant gliomas. However, the efficacy of this therapeutic modality is often limited by the occurrence of radioresistance, reflected as a diminished susceptibility of the irradiated cells to undergo cell death. Thus, cells have evolved an elegant system in response to ionizing radiation induced DNA damage, where p53, Hsp70 and/or EGFr may play an important role in the process. In the present study, we investigated whether the content of p53, Hsp70 and EGFr are associated to glioblastoma (GBM) cell radioresistance. Spheroids from U-87MG and MO59J cell lines as well as spheroids derived from primary culture of tumor tissue of one GBM patient (UGBM1) were irradiated (5, 10 and 20 Gy), their relative radioresistance were established and the p53, Hsp70 and EGFr contents were immunohistochemically determined. Moreover, we investigated whether EGFr-phospho-Akt and EGFr-MEK-ERK pathways can induce GBM radioresistance using inhibitors of activation of ERK (PD098059) and Akt (wortmannin). At 5 Gy irradiation UGBM1 and U-87MG spheroids showed growth inhibition whereas the MO59J spheroid was relatively radioresistant. Overall, no significant changes in p53 and Hsp70 expression were found following 5 Gy irradiation treatment in all spheroids studied. The only difference observed in Hsp70 content was the periphery distribution in MO59J spheroids. However, 5 Gy treatment induced a significant increase on the EGFr levels in MO59J spheroids. Furthermore, treatment with inhibitors of activation of ERK (PD098059) and Akt (wortmannin) leads to radiosensitization of MO59J spheroids. These results indicate that the PI3K-Akt and MEK-ERK pathways triggered by EGFr confer GBM radioresistance

  17. Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

    Directory of Open Access Journals (Sweden)

    Marinela Contreras

    2017-07-01

    Full Text Available Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4 and Heat shock protein 70 (HSP70 were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

  18. The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells.

    Science.gov (United States)

    Schaefer, Susanne; Svenstrup, Tina H; Guerra, Barbara

    2017-01-01

    Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells.

  19. Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens

    Science.gov (United States)

    Brokordt, Katherina B.; González, Roxana C.; Farías, William J.; Winkler, Federico M.

    2015-01-01

    Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs) are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h 2) and the additive genetic coefficient of variation (CV A) of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes) and extracellular (serum) protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R), the h 2 and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h 2 and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CV A of 13.3–16.2% in young adults and of 2.7–8.1% in pre-harvest adults. Thus, despite its low h 2, HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals. PMID

  20. Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens.

    Directory of Open Access Journals (Sweden)

    Katherina B Brokordt

    Full Text Available Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h2 and the additive genetic coefficient of variation (CVA of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes and extracellular (serum protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R, the h2 and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h2 and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CVA of 13.3-16.2% in young adults and of 2.7-8.1% in pre-harvest adults. Thus, despite its low h2, HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals.

  1. Investigating the underlying mechanism of Saccharomyces cerevisiae in response to ethanol stress employing RNA-seq analysis.

    Science.gov (United States)

    Li, Ruoyun; Xiong, Guotong; Yuan, Shukun; Wu, Zufang; Miao, Yingjie; Weng, Peifang

    2017-11-03

    Saccharomyces cerevisiae has been widely used for wine fermentation and bio-fuels production. A S. cerevisiae strain Sc131 isolated from tropical fruit shows good fermentation properties and ethanol tolerance, exhibiting significant potential in Chinese bayberry wine fermentation. In this study, RNA-sequence and RT-qPCR was used to investigate the transcriptome profile of Sc131 in response to ethanol stress. Scanning Electron Microscopy were carried out to observe surface morphology of yeast cells. Totally, 937 genes were identified differential expressed, including 587 up-regulated and 350 down-regulated genes, after 4-h ethanol stress (10% v/v). Transcriptomic analysis revealed that, most genes involved in regulating filamentous growth or pseudohyphal growth were significantly up-regulated in response to ethanol stress. The complex protein quality control machineries, Hsp90/Hsp70 and Hsp104/Hsp70/Hsp40 based chaperone system combining with ubiquitin-proteasome proteolytic pathway were both activated to recognize and degrade misfolding proteins. Genes related to biosynthesis and metabolism of two well-known stress-responsive substances trehalose and ergosterol were generally up-regulated, while genes associated with amino acids biosynthesis and metabolism processes were differentially expressed. Moreover, thiamine was also important in response to ethanol stress. This research may promote the potential applications of Sc131 in the fermentation of Chinese bayberry wine.

  2. Differences in Susceptibility to Heat Stress along the Chicken Intestine and the Protective Effects of Galacto-Oligosaccharides.

    Directory of Open Access Journals (Sweden)

    Soheil Varasteh

    Full Text Available High ambient temperatures negatively affect the human well-being as well as animal welfare and production. The gastrointestinal tract is predominantly responsive to heat stress. The currently available information about the multifaceted response to heat stress within different parts of the intestine is limited, especially in avian species. Hence, this study aims to evaluate the heat stress-induced sequence of events in the intestines of chickens. Furthermore, the gut health-promoting effect of dietary galacto-oligosaccharides (GOS was investigated in these heat stress-exposed chickens. Chickens were fed a control diet or diet supplemented with 1% or 2.5% GOS (6 days prior to and during a temperature challenge for 5 days (38-39°C, 8h per day. The parameters measured in different parts of the intestines included the genes (qPCR HSF1, HSF3, HSP70, HSP90, E-cadherin, claudin-1, claudin-5, ZO-1, occludin, TLR-2, TLR-4, IL-6, IL-8, HO-1, HIF-1α and their associated proteins HSP70, HSP90 and pan-cadherin (western blots. In addition, IL-6 and IL-8 plasma concentrations were measured by ELISA. In the jejunum, HSF3, HSP70, HSP90, E-cadherin, claudin-5, ZO-1, TLR-4, IL-6 and IL-8 mRNA expression and HSP70 protein expression were increased after heat stress exposure and a more pronounced increase in gene expression was observed in ileum after heat stress exposure, and in addition HSF1, claudin-1 and HIF-1α mRNA levels were upregulated. Furthermore, the IL-8 plasma levels were decreased in chickens exposed to heat stress. Interestingly, the heat stress-related effects in the jejunum were prevented in chickens fed a GOS diet, while dietary GOS did not alter these effects in ileum. In conclusion, our results demonstrate the differences in susceptibility to heat stress along the intestine, where the most obvious modification in gene expression is observed in ileum, while dietary GOS only prevent the heat stress-related changes in jejunum.

  3. Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

    Science.gov (United States)

    Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

    1999-01-01

    We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

  4. Genome-wide analysis of heat shock proteins in C4 model, foxtail millet identifies potential candidates for crop improvement under abiotic stress.

    Science.gov (United States)

    Singh, Roshan Kumar; Jaishankar, Jananee; Muthamilarasan, Mehanathan; Shweta, Shweta; Dangi, Anand; Prasad, Manoj

    2016-09-02

    Heat shock proteins (HSPs) perform significant roles in conferring abiotic stress tolerance to crop plants. In view of this, HSPs and their encoding genes were extensively characterized in several plant species; however, understanding their structure, organization, evolution and expression profiling in a naturally stress tolerant crop is necessary to delineate their precise roles in stress-responsive molecular machinery. In this context, the present study has been performed in C4 panicoid model, foxtail millet, which resulted in identification of 20, 9, 27, 20 and 37 genes belonging to SiHSP100, SiHSP90, SiHSP70, SiHSP60 and SisHSP families, respectively. Comprehensive in silico characterization of these genes followed by their expression profiling in response to dehydration, heat, salinity and cold stresses in foxtail millet cultivars contrastingly differing in stress tolerance revealed significant upregulation of several genes in tolerant cultivar. SisHSP-27 showed substantial higher expression in response to heat stress in tolerant cultivar, and its over-expression in yeast system conferred tolerance to several abiotic stresses. Methylation analysis of SiHSP genes suggested that, in susceptible cultivar, higher levels of methylation might be the reason for reduced expression of these genes during stress. Altogether, the study provides novel clues on the role of HSPs in conferring stress tolerance.

  5. Fisetin, a dietary flavonoid, induces apoptosis of cancer cells by inhibiting HSF1 activity through blocking its binding to the hsp70 promoter.

    Science.gov (United States)

    Kim, Joo Ae; Lee, Somyoung; Kim, Da-Eun; Kim, Moonil; Kwon, Byoung-Mog; Han, Dong Cho

    2015-06-01

    Heat shock factor 1 (HSF1) is a transcription factor for heat shock proteins (HSPs) expression that enhances the survival of cancer cells exposed to various stresses. HSF1 knockout suppresses carcinogen-induced cancer induction in mice. Therefore, HSF1 is a promising therapeutic and chemopreventive target. We performed cell-based screening with a natural compound collection and identified fisetin, a dietary flavonoid, as a HSF1 inhibitor. Fisetin abolished heat shock-induced luciferase activity with an IC50 of 14 μM in HCT-116 cancer cells. The treatment of HCT-116 with fisetin inhibited proliferation with a GI50 of 23 μM. When the cells were exposed to heat shock in the presence of fisetin, the induction of HSF1 target proteins, such as HSP70, HSP27 and BAG3 (Bcl-2-associated athanogene domain 3), were inhibited. HSP70/BAG3 complexes protect cancer cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. The downregulation of HSP70/BAG3 by fisetin significantly reduced the amounts of Bcl-2, Bcl-xL and Mcl-1 proteins, subsequently inducing apoptotic cell death. Chromatin immunoprecipitation assays showed that fisetin inhibited HSF1 activity by blocking the binding of HSF1 to the hsp70 promoter. Intraperitoneal treatment of nude mice with fisetin at 30mg/kg resulted in a 35.7% (P < 0.001) inhibition of tumor growth. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Detection of 70 kDa heat shock protein in the saliva of dairy cows.

    Science.gov (United States)

    Lamy, Elsa; Jurkovich, Viktor; Rodrigues, Lénia; Geraldo, Ana; Cachucho, Liliana; Silva, Flávio; Matos, Catarina; Capela E Silva, Fernando; Pinheiro, Cristina; Könyves, László; Bakony, Mikolt; Pereira, Alfredo

    2017-08-01

    This Research Communication describes, for the first time, the detection of HSP70 in saliva of dairy cows. Thermal stress is a major environmental stress that limits animal growth, metabolism, and productivity. The cellular response to heat stress involves the synthesis of heat shock proteins (HSPs), presumably to protect the functional stability of cells at increasing temperatures. HSP70 has been found to be present in cattle blood serum and may also be present in other secretory fluids, such as saliva, as already observed in humans. The aim of this study was to detect heat shock protein HSP70 in bovine saliva. Saliva samples were taken from higher- (n = 5) and lower milk producing (n = 5) Holstein-Friesian cows in summer and in winter for the detection of HSP70. HSP70 concentrations were assayed using the ELISA technique. Salivary HSP70 concentrations ranged from 0·524 to 12·174 ng/ml in cows. Higher salivary HSP70 concentrations were significantly associated with higher milk production and higher environmental temperature, but not with rectal temperature.

  7. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  8. Study of single nucleotide polymorphisms of tumour necrosis factors and HSP genes in nasopharyngeal carcinoma in North East India.

    Science.gov (United States)

    Lakhanpal, Meena; Singh, Laishram Chandreshwor; Rahman, Tashnin; Sharma, Jagnnath; Singh, M Madhumangal; Kataki, Amal Chandra; Verma, Saurabh; Pandrangi, Santhi Latha; Singh, Y Mohan; Wajid, Saima; Kapur, Sujala; Saxena, Sunita

    2016-01-01

    Nasopharyngeal carcinoma (NPC) is an epithelial tumour with a distinctive racial and geographical distribution. High incidence of NPC has been reported from China, Southeast Asia, and northeast (NE) region of India. The immune mechanism plays incredibly role in pathogenesis of NPC. Tumour necrosis factors (TNFs) and heat shock protein 70 (HSP 70) constitute significant components of innate as well as adaptive host immunity. Multi-analytical approaches including logistic regression (LR), classification and regression tree (CART) and multifactor dimensionality reduction (MDR) were applied in 120 NPC cases and 100 controls to explore high order interactions among TNF-α (-308 G>A), TNF β (+252 A>G), HSP 70-1 (+190 G>C), HSP 70-hom (+2437 T>C) genes and environmental risk factors. TNF β was identified as the primary etiological factor by all three analytical approaches. Individual analysis of results showed protective effect of TNF β GG genotype (adjusted odds ratio (OR2) = 0.27, 95 % CI = 0.125-0.611, P = 0.001), HSP 70 (+2437) CC genotype (OR2 = 0.17, 95 % CI = 0.0430.69, P = 0.013), while AG genotype of TNF β was found significantly associated with risk of NPC (OR2 = 1.97, 95 % CI = 1.019-3.83, P = 0.04). Analysis of environmental factors demonstrated association of alcohol consumption, living in mud houses and use of firewood for cooking as major risk factors for NPC. Individual haplotype association analysis showed significant risk associated with GTGA haplotype (OR = 68.61, 95 % CI = 2.47-190.37, P = 0.013) while a protective effect with CCAA and GCGA haplotypes (OR = 0.19, 95 % CI = 0.05-0.75, P = 0.019; OR = 0.01 95 % CI = 0.05-0.30, P = 0.007). The multi-analytical approaches applied in this study helped in identification of distinct gene-gene and gene-environment interactions significant in risk assessment of NPC.

  9. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

    International Nuclear Information System (INIS)

    Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

    1998-01-01

    Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

  10. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

    Science.gov (United States)

    Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

    1991-01-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

  11. Non-lethal heat shock increased Hsp70 and immune protein transcripts but not Vibrio tolerance in the white-leg shrimp.

    Directory of Open Access Journals (Sweden)

    Nguyen Hong Loc

    Full Text Available Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70 and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO, peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined.

  12. Non-Lethal Heat Shock Increased Hsp70 and Immune Protein Transcripts but Not Vibrio Tolerance in the White-Leg Shrimp

    Science.gov (United States)

    Loc, Nguyen Hong; MacRae, Thomas H.; Musa, Najiah; Bin Abdullah, Muhd Danish Daniel; Abdul Wahid, Mohd. Effendy; Sung, Yeong Yik

    2013-01-01

    Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70) and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO), peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined. PMID:24039886

  13. Differential expression analysis of boron transporters and some stress-related genes in response to 24-epibrassinolide and boron by semi-quantitative RT-PCR in Arabidopsis thaliana (L. Heynh

    Directory of Open Access Journals (Sweden)

    Surgun Yonca

    2016-01-01

    Full Text Available Plant steroidal hormones, brassinosteroids (BRs, promote plant developmental processes and enhance tolerance to several abiotic stresses including high boron (B stress. To examine the possible role of BR in high B-induced stress at the transcriptional level, we investigated the response of B transporter genes (BOR1-4, high B-induced genes (MATE, Hsp-like, BR-induced genes (Hsp70-4, Hsp90-1 and other stress-related genes (LTI/COR78, LEA4-5 upon exogenous treatments of 24-epibrassinolide (EBL on Arabidopsis thaliana (L. Heynh exposed to high concentrations of boric acid (BA using semi-quantitative RT-PCR. BA treatments led to down regulation of BOR1 and BOR3 genes in leaf and root tissues and higher concentration of EBL further decreased expression of these genes in roots. The expression of high B-induced genes was observed to be upregulated by 1 μM EBL treatment under high B stress in both tissues of the seedlings. The upregulation of BR-induced genes were clearly evident in root tissues co-treated with 1 μM EBL and BA as compared to BA alone. Higher concentration of EBL was found to be more effective in increasing expression of LTI/COR78 gene in root and LEA4-5 gene in shoot tissues. To our knowledge, this is the first report how exogenous application of EBL modulates high B stress responses at molecular level in model plant Arabidopsis thaliana.

  14. Ultraviolet filters differentially impact the expression of key endocrine and stress genes in embryos and larvae of Chironomus riparius.

    Science.gov (United States)

    Ozáez, Irene; Morcillo, Gloria; Martínez-Guitarte, José-Luis

    2016-07-01

    Several organic UV filters have hormonal activity in vertebrates, as demonstrated in fishes, rodents and human cells. Despite the accumulation of filter contaminants in aquatic systems, research on their effects on the endocrine systems of freshwaters invertebrates is scarce. In this work, the effects of five frequently used UV filters were investigated in embryos and larvae of Chironomus riparius, which is a reference organism in ecotoxicology. LC50 values for larvae as well as the percentage of eclosion of eggs were determined following exposures to: octyl-p-methoxycinnamate (OMC) also known as 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4MBC); 4-hydroxybenzophenone (4HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). To assess sublethal effects, expression levels of the genes coding for the ecdysone receptor (EcR) and heat shock protein HSP70 were investigated as biomarkers for endocrine and stress effects at the cellular level. Life-stage-dependent sensitivity was found. In embryos, all of the UV filters provoked a significant overexpression of EcR at 24h after exposure. OC, 4MBC and OD-PABA also triggered transcriptional activation of the hsp70 stress gene in embryos. In contrast, in larvae, only 4MBC and OMC/EHMC increased EcR and hsp70 mRNA levels and OD-PABA upregulated only the EcR gene. These results revealed that embryos are particularly sensitive to UV filters, which affect endocrine regulation during development. Most UV filters also triggered the cellular stress response, and thus exhibit proteotoxic effects. The differences observed between embryos and larvae and the higher sensitivity of embryos highlight the importance of considering different life stages when evaluating the environmental risks of pollutants, particularly when analyzing endocrine effects. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

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    Sanjeev Gupta

    2010-07-01

    Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

  16. Durum wheat diversity for heat stress tolerance during inflorescence emergence is correlated to TdHSP101C expression in early developmental stages.

    Directory of Open Access Journals (Sweden)

    Miguel Bento

    Full Text Available The predicted world population increase along with climate changes threatens sustainable agricultural supply in the coming decades. It is therefore vital to understand crops diversity associated to abiotic stress response. Heat stress is considered one of the major constrains on crops productivity thus it is essential to develop new approaches for a precocious and rigorous evaluation of varietal diversity regarding heat tolerance. Plant cell membrane thermostability (CMS is a widely used method for wheat thermotolerance assessment although its limitations require complementary solutions. In this work we used CMS assay and explored TdHSP101C genes as an additional tool for durum wheat screening. Genomic and transcriptomic analyses of TdHSP101C genes were performed in varieties with contrasting CMS results and further correlated with heat stress tolerance during fertilization and seed development. Although the durum wheat varieties studied presented a very high homology on TdHSP101C genes (>99% the transcriptomic assessment allowed the discrimination between varieties with good CMS results and its correlation with differential impacts of heat treatment during inflorescence emergence and seed development on grain yield. The evidences here reported indicate that TdHSP101C transcription levels induced by heat stress in fully expanded leaves may be a promising complementary screening tool to discriminate between durum wheat varieties identified as thermotolerant through CMS.

  17. Hsp90 Is Essential under Heat Stress in the Bacterium Shewanella oneidensis

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    Flora Ambre Honoré

    2017-04-01

    Full Text Available The Hsp90 chaperone is essential in eukaryotes and activates a large array of client proteins. In contrast, its role is still elusive in bacteria, and only a few Hsp90 bacterial clients are known. Here, we found that Hsp90 is essential in the model bacterium Shewanella oneidensis under heat stress. A genetic screen for Hsp90 client proteins identified TilS, an essential protein involved in tRNA maturation. Overexpression of TilS rescued the growth defect of the hsp90 deletion strain under heat stress. In vivo, the activity and the amount of TilS were significantly reduced in the absence of Hsp90 at high temperature. Furthermore, we showed that Hsp90 interacts with TilS, and Hsp90 prevents TilS aggregation in vitro at high temperature. Together, our results indicate that TilS is a client of Hsp90 in S. oneidensis. Therefore, our study links the essentiality of bacterial Hsp90 at high temperature with the identification of a client.

  18. Resistência à Síndrome Ascítica, Competência Homeotérmica e Níveis de Hsp70 no Coração e Pulmão de Frangos de Corte Resistance to Ascites Syndrome, Homoeothermic Competence and Levels of Hsp70 in the Heart and Lung of Broilers

    Directory of Open Access Journals (Sweden)

    Renata Hernandes

    2002-06-01

    Full Text Available Como em outros seres vivos, também nas células das aves ocorre a síntese das proteínas de baixo peso molecular (Hsp, cujo aumento é induzido sob condições de estresse. As Hsps têm um papel importante na manutenção da integridade celular, questiona-se o seu envolvimento no mecanismo de proteção celular de órgãos alvos na ocorrência da síndrome ascítica (SA. Este trabalho objetivou avaliar a temperatura corporal e os níveis da Hsp70 no coração e pulmão de frangos de corte Hubbard (sensível à SA e caipira de pescoço-pelado (resistente, criados em termoneutralidade (25°C e frio (16°C entre 10 e 45 dias de idade. Foram utilizados 192 pintos machos, 96 de cada linhagem. Não houve mortalidade por SA nas aves caipiras. Nas aves Hubbard, a mortalidade devida à SA foi de 4% e 41% em ambiente termoneutro e frio, respectivamente. Em ambiente frio, a temperatura corporal das aves Hubbard foi menor que a das caipiras. A temperatura corporal e o nível de Hsp70 do coração das aves Hubbard diminuíram com o aumento da idade, mas não nas aves caipiras, os quais se mantiveram constantes, inclusive a Hsp70 do pulmão. Independente da idade ou da temperatura, o nível de Hsp70 no pulmão das aves caipiras era superior ao das aves Hubbard. Em relação às aves Hubbard, as caipiras são homeotérmicas mais competentes e apresentam uma maior indução de Hsp70 nos órgãos primariamente afetados na SA, mas este não parece ser o sistema de proteção contra SA, a qual as aves de pescoço pelado são resistentes.Similar to other living animals, the cells of the birds also synthesize small proteins (heat shock protein, Hsp, which increasing levels can be induce by stress. The Hsp have a relevant function in maintaining the integrity of the cell, and we question if they are involved in the mechanism of the cellular protection of target organs affected by ascites syndrome (AS. The objective of this study was to evaluate the body temperature

  19. Substrate Discrimination by ClpB and Hsp104

    Directory of Open Access Journals (Sweden)

    Danielle M. Johnston

    2017-05-01

    Full Text Available ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

  20. The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

    Science.gov (United States)

    Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

    2004-05-21

    The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

  1. Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

    Directory of Open Access Journals (Sweden)

    Li Y

    2016-07-01

    Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

  2. In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

    Science.gov (United States)

    de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

    2013-02-01

    Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

  3. Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: Influence of elevated dietary iron

    Energy Technology Data Exchange (ETDEWEB)

    Kwong, Raymond W.M. [Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3 (Canada); Andres, Jose A. [Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E2 (Canada); Niyogi, Som, E-mail: som.niyogi@usask.ca [Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E2 (Canada)

    2011-03-15

    Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine > kidney > stomach > liver > gill > carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.

  4. Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: Influence of elevated dietary iron

    International Nuclear Information System (INIS)

    Kwong, Raymond W.M.; Andres, Jose A.; Niyogi, Som

    2011-01-01

    Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine > kidney > stomach > liver > gill > carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.

  5. Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

    NARCIS (Netherlands)

    Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

    We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

  6. Food odor, visual danger stimulus, and retrieval of an aversive memory trigger heat shock protein HSP70 expression in the olfactory lobe of the crab Chasmagnathus granulatus.

    Science.gov (United States)

    Frenkel, L; Dimant, B; Suárez, L D; Portiansky, E L; Delorenzi, A

    2012-01-10

    Although some of the neuronal substrates that support memory process have been shown in optic ganglia, the brain areas activated by memory process are still unknown in crustaceans. Heat shock proteins (HSPs) are synthesized in the CNS not only in response to traumas but also after changes in metabolic activity triggered by the processing of different types of sensory information. Indeed, the expression of citosolic/nuclear forms of HSP70 (HSC/HSP70) has been repeatedly used as a marker for increases in neural metabolic activity in several processes, including psychophysiological stress, fear conditioning, and spatial learning in vertebrates. Previously, we have shown that, in the crab Chasmagnathus, two different environmental challenges, water deprivation and heat shock, trigger a rise in the number of glomeruli of the olfactory lobes (OLs) expressing HSC/HSP70. In this study, we initially performed a morphometric analysis and identified a total of 154 glomeruli in each OL of Chasmagnathus. Here, we found that crabs exposed to food odor stimuli also showed a significant rise in the number of olfactory glomeruli expressing HSC/HSP70. In the crab Chasmagnathus, a powerful memory paradigm based on a change in its defensive strategy against a visual danger stimulus (VDS) has been extensively studied. Remarkably, the iterative presentation of a VDS caused an increase as well. This increase was triggered in animals visually stimulated using protocols that either build up a long-term memory or generate only short-term habituation. Besides, memory reactivation was sufficient to trigger the increase in HSC/HSP70 expression in the OL. Present and previous results strongly suggest that, directly or indirectly, an increase in arousal is a sufficient condition to bring about an increase in HSC/HSP70 expression in the OL of Chasmagnathus. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Monitoring the Induction of Heat Shock Factor 1/Heat Shock Protein 70 Expression following 17-Allylamino-Demethoxygeldanamycin Treatment by Positron Emission Tomography and Optical Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Mikhail Doubrovin

    2012-01-01

    Full Text Available The cell response to proteotoxic cell stresses is mediated primarily through activation of heat shock factor 1 (HSF1. This transcription factor plays a major role in the regulation of the heat shock proteins (HSPs, including HSP70. We demonstrate that an [124I]iodide-pQHNIG70 positron emission tomography (PET reporter system that includes an inducible HSP70 promoter can be used to image and monitor the activation of the HSF1/HSP70 transcription factor in response to drug treatment (17-allylamino-demethoxygeldanamycin [17-AAG]. We developed a dual imaging reporter (pQHNIG70 for noninvasive imaging of the heat shock response in cell culture and living animals previously and now study HSF1/HSP70 reporter activation in both cell culture and tumor-bearing animals following exposure to 17-AAG. 17-AAG (10–1,000 nM induced reporter expression; a 23-fold increase was observed by 60 hours. Good correspondence between reporter expression and HSP70 protein levels were observed. MicroPET imaging based on [124I]iodide accumulation in pQHNIG70-transduced RG2 xenografts showed a significant 6.2-fold reporter response to 17-AAG, with a corresponding increase in tumor HSP70 and in tumor human sodium iodide symporter and green fluorescent protein reporter proteins. The HSF1 reporter system can be used to screen anticancer drugs for induction of cytotoxic stress and HSF1 activation both in vitro and in vivo.

  8. Whey protein hydrolysate enhances HSP90 but does not alter HSP60 and HSP25 in skeletal muscle of rats.

    Directory of Open Access Journals (Sweden)

    Carolina Soares Moura

    Full Text Available Whey protein hydrolysate (WPH intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed and exercised (stressed groups, and were fed with three different sources of protein: whey protein (WP, whey protein hydrolysate (WPH and casein (CAS as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85, GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.

  9. Hsp27, Hsp70, and metallothionein in MDCK and LLC-PK1 renal epithelial cells: effects of prolonged exposure to cadmium

    International Nuclear Information System (INIS)

    Bonham, Rita T.; Fine, Michael R.; Pollock, Fiona M.; Shelden, Eric A.

    2003-01-01

    Cadmium is a widely distributed industrial and environmental toxin. The principal target organ of chronic sublethal cadmium exposure is the kidney. In renal epithelial cells, acute high-dose cadmium exposure induces differential expression of proteins, including heat shock proteins. However, few studies have examined heat shock protein expression in cells after prolonged exposure to cadmium at sublethal concentrations. Here, we assayed total cell protein, neutral red uptake, cell death, and levels of metallothionein and heat shock proteins Hsp27 and inducible Hsp70 in cultures of MDCK and LLC-PK1 renal epithelial cells treated with cadmium for 3 days. Treatment with cadmium at concentrations equal to or greater than 10 μM (LLC-PK1) or 25 μM (MDCK) reduced measures of cell vitality and induced cell death. However, a concentration-dependent increase in Hsp27 was detected in both cell types treated with as little as 5 μM cadmium. Accumulation of Hsp70 was correlated only with cadmium treatment at concentrations also causing cell death. Metallothionein was maximally detected in cells treated with cadmium at concentrations that did not reduce cell vitality, and further increases were not detected at greater concentrations. These results reveal that heat shock proteins accumulate in renal epithelial cells during prolonged cadmium exposure, that cadmium induces differential expression of heat shock protein in epithelial cells, and that protein expression patterns in epithelial cells are specific to the cadmium concentration and degree of cellular injury. A potential role for Hsp27 in the cellular response to sublethal cadmium-induced injury is also implicated by our results

  10. Resveratrol induces antioxidant and heat shock protein mRNA expression in response to heat stress in black-boned chickens.

    Science.gov (United States)

    Liu, L L; He, J H; Xie, H B; Yang, Y S; Li, J C; Zou, Y

    2014-01-01

    This study investigated the effects of dietary resveratrol at 0, 200, 400, or 600 mg/kg of diet on the performance, immune organ growth index, serum parameters, and expression levels of heat shock protein (Hsp) 27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius, thymus, and spleen of 42-d-old female black-boned chickens exposed to heat stress at 37 ± 2°C for 15 d. The results showed that heat stress reduced daily feed intake and BW gain; decreased serum glutathione (GSH), growth hormone, and insulin-like growth factor-1 levels; and inhibited GSH peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) activities compared with birds subjected to thermo-neutral circumstances. Chickens that were fed diets supplemented with resveratrol exhibited a linear increase in feed intake and BW gain (P stress. In contrast, serum malonaldehyde concentrations were decreased (P stress also reduced (P stress and coincided with an increase in supplemental resveratrol levels. The expression of Hsp27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius and spleen were increased (P stress compared with no heat stress. Resveratrol attenuated the heat stress-induced overexpression of Hsp27, Hsp70, and Hsp90 mRNA in the bursa of Fabricius and spleen and increased the low expression of Hsp27 and Hsp90 mRNA in thymus upon heat stress. The results suggest that supplemental resveratrol improves growth performance and reduces oxidative stress in heat-stressed black-boned chickens by increasing serum growth hormone concentrations and modulating the expression of heat shock genes in organs of the immune system.

  11. Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

    Science.gov (United States)

    Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

    2016-11-01

    Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

  12. Microbiota Composition, HSP70 and Caspase-3 Expression as Marker for Colorectal Cancer Patients in Aceh, Indonesia

    Directory of Open Access Journals (Sweden)

    Fauzi Yusuf

    2017-02-01

    Full Text Available Aim: to investigate the relationship between microbiota composition with HSP70 and Caspase-3 expressions in colon tissue as an initial study to develop the candidate for early detection of colorectal cancer for Indonesian patients. Methods: this is a cross-sectional study on 32 patients undergoing colonoscopy; 16 patients of colorectal cancer (CRC while the other 16 patients are not (colitis and internal hemorrhoid. The composition of microbiota in stool samples was examined using 16S rRNA Denaturing Gradient Gel Electrophoresis (DDGE while expression of HSP70 was examined by immunohistochemistry and Caspase-3 by using Haematoxylin-Eosin(HE staining to determine the morphological changes in colon tissue. Results: analysis of PCR-DDGE shows a different composition of microbiota between patients with CRC and non-CRC. All CRC patients showed disappearance of dominant band from Bifidobacterium groups. Histological observation based on Inter Class Correlation (ICC test from all slide showed a high scores (5.2-9.2 in CRC patients and low scores (1.7-2.4 in non-CRC patients. HSP70 expression was increased significantly in CRC patients with the highest percentage of 84%, while expression of caspase-3 decreased with the highest percentage of 21%. Statistical analysis showed that the incidence of colorectal cancer was associated with the expression of HSP 70 (p<0.001, and Caspase 3 (p<0.001. Conclusion: bifidobacterium is an important indicator for colorectal cancer patients that show disappearance of dominant band, while expression of HSP70 increased and the Caspase-3 expression decreased significantly.

  13. Effects of heavy metals on ultrastructure and Hsp70 induction in Lemna minor L. exposed to water along the Sarno River, Italy.

    Science.gov (United States)

    Basile, A; Sorbo, S; Cardi, M; Lentini, M; Castiglia, D; Cianciullo, P; Conte, B; Loppi, S; Esposito, S

    2015-04-01

    The effects of freshwater pollution in the highly contaminated river Sarno (Campania, Southern Italy) have been evaluated using bags containing the aquatic plant Lemna minor (Lemnacee, Arales), in order to determine morpho-physiological modifications as a response to pollutants. The exposition of Lemna bags for 7 days on three different sites along the river path showed alterations in chloroplasts and vacuoles shape and organization. Moreover, some specimens were exposed in vitro at the same heavy metal (HM) concentrations measured in the polluted sites of the river, and compared with data from the bag experiment; to verify the dose and time dependent effects, samples were exposed to HM in vitro at concentrations ranging from 10(-6) to 10(-4)M up to 7 days. Transmission electron microscopy (TEM) observations on in vitro plants confirmed that ultrastructural alterations affected most of plastids and the shape of different subcellular structures, namely vacuoles; in in vitro stressed specimens, Heat Shock Proteins 70 (Hsp70) levels changed, in dependence of changing levels of HM measured in different sites along the river path. Thus L. minor exhibited a possible correlation between the levels of HM pollution and Hsp70 occurrence; interestingly, the data presented showed that copper specifically increased Hsp70 levels at concentrations detected in polluted river waters, whereas cadmium and lead did not; on the other side, the latter represent highly toxic elements when specimens were exposed to higher levels in vitro. The effects of specific elements in vitro are compared to those observed in bags exposed along the river path; thus results are examined in order to propose L. minor as an organism able to be utilized to monitor heavy metals pollution; the possibility of using Hsp70s as specific markers of HM pollution is discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Antileukemic activity of the HSP70 inhibitor pifithrin-μ in acute leukemia

    International Nuclear Information System (INIS)

    Kaiser, M; Kühnl, A; Reins, J; Fischer, S; Ortiz-Tanchez, J; Schlee, C; Mochmann, L H; Heesch, S; Benlasfer, O; Hofmann, W-K; Thiel, E; Baldus, C D

    2011-01-01

    Heat shock protein (HSP) 70 is aberrantly expressed in different malignancies and has emerged as a promising new target for anticancer therapy. Here, we analyzed the in vitro antileukemic effects of pifithrin-μ (PFT-μ), an inhibitor of inducible HSP70, in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines, as well as in primary AML blasts. PFT-μ significantly inhibited cell viability at low micromolar concentrations in all cell lines tested, with IC50 values ranging from 2.5 to 12.7 μ, and was highly active in primary AML blasts with a median IC50 of 8.9 μ (range 5.7–37.2). Importantly, higher IC50 values were seen in normal hematopoietic cells. In AML and ALL, PFT-μ induced apoptosis and cell cycle arrest in a dose-dependent fashion. PFT-μ also led to an increase of the active form of caspase-3 and reduced the intracellular concentrations of AKT and ERK1/2 in NALM-6 cells. Moreover, PFT-μ enhanced cytotoxicity of cytarabine, 17-(allylamino)-17-desmethoxygeldanamycin, suberoylanilide hydroxamic acid, and sorafenib in NALM-6, TOM-1 and KG-1a cells. This is the first study demonstrating significant antileukemic effects of the HSP70 inhibitor PFT-μ, alone and in combination with different antineoplastic drugs in both AML and ALL. Our results suggest a potential therapeutic role for PFT-μ in acute leukemias

  15. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

    DEFF Research Database (Denmark)

    Rohde, Mikkel; Daugaard, Mads; Jensen, Mette Hartvig

    2005-01-01

    Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70...... proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas...

  16. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    Science.gov (United States)

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  17. [Antiarrhythmic effect of oligonucleotides accompanied by activation of HSP70 protein in the heart of rats].

    Science.gov (United States)

    Kruglov, S V; Terekhina, O L; Smirnova, E A; Kashaeva, O V; Belkina, L M

    2015-01-01

    The mechanisms of the protective effect of oligonucleotides (OGN) during pathological processes are poorlyunderstood. The goal of this work was to study the effect of OGN on arrhythmias induced by myocardial ischemia and reperfusion, and the HSP70 level in the heart. As a source of OGN was used the drug "Derinat" ("Technomedservis", Russia). In male Wistar rats were pre-treated the drug for 7 days (i/m, 7.5 mg/kg).The intensity of the arrhythmias was assessed by ECG during 10 min occlusion of the left coronary artery and subsequent 5 min of reperfusion. Protein HSP70 determined in the left ventricle of the heart by Western-blot analysis. During ischemia, this drug reduced duration of extrasystolia by 13 times and the incidence of ventricular tachycardia by 1.5 times. During reperfusion the drug reduced the incidence of ventricular fibrillation, a more than 2-fold, as compared with the control (respectively 23% vs 56%) and by 5 times its duration (8,4 ± 2,3 48,1 ± sec vs 18 7 sec). "Derinat" increased the HSP70 level in the heart by 65% compared with control. These data support the fact that the activation of HSP70 synthesis, induced by OGN is one of the mechanisms that increases the heart resistance to the ischemic and reperfusion damages.

  18. Stress memory induced rearrangements of HSP transcription, photosystem II photochemistry and metabolism of tall fescue (Festuca arundinacea Schreb. in response to high-temperature stress

    Directory of Open Access Journals (Sweden)

    Tao eHu

    2015-06-01

    Full Text Available When plants are pre-exposed to stress, they can produce some stable signals and physiological reactions that may be carried forward as ‘stress memory’. However, there is insufficient information about is known about plants’ stress memory responses mechanisms. Here, two tall fescue genotypes, heat-tolerant PI 574522 and heat-sensitive PI 512315, were subjected to recurring high-temperature pre-acclimation treatment. Two heat shock protein (HSP genes, LMW-HSP and HMW-HSP, exhibited transcriptional memory for their higher transcript abundance during one or more subsequent stresses (S2, S3, S4 relative to the first stress (S1, and basal transcript levels during the recovery states (R1, R2 and R3. Activated transcriptional memory from two trainable genes could persist up to 4 days, and induce higher thermotolerance in tall fescue. This was confirmed by greater turf quality and lower electrolyte leakage. Pre-acclimation treatment inhibited the decline at steps of O-J-I-P and energy transport fluxes in active Photosystem II reaction center (PSII RC for both tall fescue genotypes. The heat stress memory was associated with major shifts in leaf metabolite profiles. Furthermore, there was an exclusive increase in leaf organic acids (citric acid, malic acid, tris phosphoric acid, threonic acid, sugars (sucrose, glucose, idose, allose, talose, glucoheptose, tagatose, psicose, amino acids (serine, proline, pyroglutamic acid, glycine, alanine and one fatty acid (butanoic acid in pre-acclimated plants. These discoveries involved in transcriptional memory, PSII RC energy transport and metabolite profiles could provide new insights into the plant high–temperature response process.

  19. Feeding glycerol-enriched yeast culture improves performance, energy status, and heat shock protein gene expression of lactating Holstein cows under heat stress.

    Science.gov (United States)

    Liu, J; Ye, G; Zhou, Y; Liu, Y; Zhao, L; Liu, Y; Chen, X; Huang, D; Liao, S F; Huang, K

    2014-06-01

    This study was conducted to evaluate the effects of supplemental common yeast culture (CY) and glycerol-enriched yeast culture (GY) on performance, plasma metabolites, antioxidant status, and heat shock protein 70 (HSP70) mRNA expression in lactating Holstein cows under heat stress. During summer months, 30 healthy multiparous lactating cows (parity 3.25 ± 0.48; 60 ± 13 d in milk [DIM]; 648 ± 57 kg BW; an average milk yield of 33.8 ± 1.6 kg/d) were blocked by parity, previous milk yield, and DIM and randomly allocated to 3 dietary treatments: no supplemental yeast culture (Control), 1 L/d of CY (33.1 g yeast) per cow, and 2 L/d of GY (153.2 g glycerol and 31.6 g yeast) per cow. During the 60-d experiment, values of air temperature and relative humidity inside the barn were recorded hourly every 3 d to calculate temperature-humidity index (THI). Weekly rectal temperatures (RT) and respiration rates and daily DMI and milk yield were recorded for all cows. Milk and blood samples were taken twice monthly, and BW and BCS were obtained on d 0 and 60. In this experiment, THI values indicated cows experienced a moderate heat stress. Cows supplemented with CY and GY had greater yields of milk, energy-corrected milk and milk fat, and milk fat percent but lower HSP70 mRNA expression in peripheral blood lymphocytes than Control cows (P cows. In conclusion, either CY or GY supplementation partially mitigated the negative effects of heat stress on performance and HSP70 mRNA expression of lactating cows, and GY supplementation provided additional improvements in energy status and HSP70 gene expression of lactating cows.

  20. Expression of heat shock proteins (HSPs) in Aedes aegypti (L) and Aedes albopictus (Skuse) (Diptera: Culicidae) larvae in response to thermal stress.

    Science.gov (United States)

    Sivan, Arun; Shriram, Ananganallur Nagarajan; Muruganandam, Nagarajan; Thamizhmani, Ramanathan

    2017-03-01

    Climatic changes are responsible, to a certain extent for the occurrence and spread of arboviral pathogens world over. Temperature is one of the important abiotic factors influencing the physiological processes of mosquitoes. Several genes of heat shock protein (HSP) families are known to be expressed in mosquitoes, which aid in overcoming stress induced by elevated temperature. In order to understand expression of HSP family genes in the Andaman population of Aedes aegypti and Aedes albopictus, we used quantitative real-time polymerase chain reaction (qPCR) to examine expression levels of HSPs in response to thermal stress under laboratory and in actual field conditions. HSP genes AeaHsp26, AeaHsp83 and AeaHsc70 were examined by comparing relative transcript expression levels at 31°C, 33°C, 34°C, 37°C and 39°C respectively. Enhanced up-regulation of HSPs was evident in third instar larvae of Ae. aegypti with rise in water temperatures (31°C, 33°C, 34°C) in the containers in the nature and thermally stressed (37°C and 39°C) in laboratory conditions. In Ae. albopictus up-regulation of HSPs was observed in field conditions at 34°C only and when thermally treated at 37°C, while down regulation was evident in larvae subjected to thermal stress in laboratory at 39°C. Data on expression levels revealed that larvae of Ae. aegypti was tolerant to thermal stress, while Ae. albopictus larvae was sensitive to heat shock treatment. Statistical analysis indicated that AeaHsp83 genes were significantly up-regulated in Ae. aegypti larvae after 360min exposure to high temperature (39°C). The difference in expression levels of AeaHsp26, AeaHsc70 and AeaHsp83 genes in Ae. albopictus larvae was statistically significant between different exposure temperatures. All of these genes were significantly up-regulated at 37°C. These results indicate that AeaHsp26, AeaHsc70 and AeaHsp83 are important markers of stress and perhaps function as proteins conferring protection and

  1. Effect of acute exposure to cadmium on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic midge Chironomus riparius

    International Nuclear Information System (INIS)

    Planello, R.; Martinez-Guitarte, J.L.; Morcillo, G.

    2010-01-01

    Cadmium is a widespread and highly toxic pollutant of particular ecotoxicological relevance for aquatic ecosystems where it accumulates. To identify biomarkers for ecotoxicity monitoring, the effect of cadmium on the expression of different genes related to the stress response as well as to the ecdysone hormone-signalling pathway was studied in the aquatic larvae of Chironomus riparius (Diptera, Chironomidae), a standard test organism in aquatic toxicology testing. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to evaluate the effects of acute and short-term cadmium exposures (10 mM CdCl 2 , 12 h and 24 h) on the expression of hsp70, hsc70, hsp90 and hsp40 genes, as well as on that of the ecdysone hormonal-receptor genes (EcR and usp). A significant 3-fold increase in the level of hsp70 gene transcripts was induced by the treatment, whereas neither the other stress genes tested (hsp90 and hsp40) nor the constitutive form of hsp70, hsc70, was affected in the larvae exposed to cadmium. These results show that hsp70 is differentially activated to other environmentally regulated heat-shock genes, and constitutes a biomarker of exposure to this toxic metal. In addition, we also found that cadmium is able to alter the expression of the ecdysone receptor gene (EcR), whose mRNA level is significantly increased whereas usp levels remained unaltered. This finding, evidenced for the first time in invertebrates, supports the view that cadmium has the ability to mimic the effect of the hormone by the activation of the ecdysone nuclear receptor, which may partly explain the endocrine disruption capability that has been previously suggested for this toxic metal. Our research adds to the growing evidence implicating heavy metals, and cadmium in particular, as potential endocrine disruptive agents and may have significant implications for ecological risk assessment of endocrine-disrupting compounds in invertebrates.

  2. Effect of acute exposure to cadmium on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic midge Chironomus riparius

    Energy Technology Data Exchange (ETDEWEB)

    Planello, R.; Martinez-Guitarte, J.L. [Grupo de Biologia y Toxicologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, UNED, Senda del Rey 9, 28040 Madrid (Spain); Morcillo, G., E-mail: gmorcillo@ccia.uned.es [Grupo de Biologia y Toxicologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, UNED, Senda del Rey 9, 28040 Madrid (Spain)

    2010-03-01

    Cadmium is a widespread and highly toxic pollutant of particular ecotoxicological relevance for aquatic ecosystems where it accumulates. To identify biomarkers for ecotoxicity monitoring, the effect of cadmium on the expression of different genes related to the stress response as well as to the ecdysone hormone-signalling pathway was studied in the aquatic larvae of Chironomus riparius (Diptera, Chironomidae), a standard test organism in aquatic toxicology testing. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to evaluate the effects of acute and short-term cadmium exposures (10 mM CdCl{sub 2}, 12 h and 24 h) on the expression of hsp70, hsc70, hsp90 and hsp40 genes, as well as on that of the ecdysone hormonal-receptor genes (EcR and usp). A significant 3-fold increase in the level of hsp70 gene transcripts was induced by the treatment, whereas neither the other stress genes tested (hsp90 and hsp40) nor the constitutive form of hsp70, hsc70, was affected in the larvae exposed to cadmium. These results show that hsp70 is differentially activated to other environmentally regulated heat-shock genes, and constitutes a biomarker of exposure to this toxic metal. In addition, we also found that cadmium is able to alter the expression of the ecdysone receptor gene (EcR), whose mRNA level is significantly increased whereas usp levels remained unaltered. This finding, evidenced for the first time in invertebrates, supports the view that cadmium has the ability to mimic the effect of the hormone by the activation of the ecdysone nuclear receptor, which may partly explain the endocrine disruption capability that has been previously suggested for this toxic metal. Our research adds to the growing evidence implicating heavy metals, and cadmium in particular, as potential endocrine disruptive agents and may have significant implications for ecological risk assessment of endocrine-disrupting compounds in invertebrates.

  3. Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity.

    Science.gov (United States)

    Périard, Julien D; Ruell, Patricia; Caillaud, Corinne; Thompson, Martin W

    2012-05-01

    Extracellular heat-shock protein 72 (eHsp72) expression during exercise-heat stress is suggested to increase with the level of hyperthermia attained, independent of the rate of heat storage. This study examined the influence of exercise at various intensities to elucidate this relationship, and investigated the association between eHsp72 and eHsp27. Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake in hot conditions (40°C, 50% RH). Core temperature, heart rate, oxidative stress, and blood lactate and glucose levels were measured to determine the predictor variables associated with eHsp expression. At exhaustion, heart rate exceeded 96% of maximum in both conditions. Core temperature reached 39.7°C in the 60% trial (58.9 min) and 39.0°C in the 75% trial (27.2 min) (P exercise may relate to the duration (i.e., core temperature attained) and intensity (i.e., rate of increase in core temperature) of exercise. Thus, the immuno-inflammatory release of eHsp72 and eHsp27 in response to exercise in the heat may be duration and intensity dependent.

  4. Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

    International Nuclear Information System (INIS)

    Bryantsev, A.L.; Chechenova, M.B.; Shelden, E.A.

    2007-01-01

    During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus

  5. Heat-shock protein (Hsp70) and cytochrome P-450 (CYP1A) in the white mullet Mugil curema (Pisces:Mugilidae) as biomarkers to assess environmental quality in coastal lagoons.

    Science.gov (United States)

    Rios-Sicairos, Julian; Betancourt-Lozano, Miguel; Leal-Tarin, Beatriz; Hernandez-Cornejo, Rubi; Aguilar-Zarate, Gabriela; Garcia-De-La-Parra, Luz Maria; Gutierrez, Jesus N; Marquez-Rocha, Facundo; Garcia-Gasca, Alejandra

    2010-01-01

    Biomarkers have been useful tools to monitor some effects of pollution in coastal environments. Hepatic expression of heat-shock protein 70 (Hsp70) and cytochrome P450 1A (CYP1A) were analyzed in white mullet (Mugil curema) by RT-PCR from July, 2005 until July, 2006 in three coastal lagoons located in the southern Gulf of California, Mexico. These three coastal systems receive contaminants derived from local anthropogenic activities. Heat-shock proteins function to maintain protein integrity in the presence of stressors (such as heat or chemicals) and can be used as biomarkers of homeostatic alterations in polluted environments, whereas cytochrome P450 family members participate in steroid hormone synthesis and metabolism, and in xenobiotic transformation as a detoxification mechanism. The expression levels of both genes showed consistency in time and space, and presented a high overall correlation (r = 0.731, P < 0.001). Regardless of a high individual variability, both genes presented higher expression levels in the Urias Estuary (P < 0.001 and P < 0.05 for CYP1A and Hsp70, respectively), which was considered the most polluted among the three systems, especially during the rainy season (summer to fall). Gene expression levels were significantly associated with non-halogenated hydrocarbon concentrations in sediments during the sampling period (r = 0.686, P = 0.019 for CYP1A and r = 0.91, P < 0.001 for Hsp70), suggesting that both genes respond to chemicals in the environment. The results indicate that Mugil curema is a good candidate species to implement biomonitoring programs in tropical coastal environments.

  6. [The impact of melafen on the expression of chloroplastic chaperone protein HSP70B and photosynthetic pigments in cells of Chlamydomonas reinhardtii].

    Science.gov (United States)

    Ermokhina, O V; Belkina, G G; Oleskina, Iu P; Fattakhov, S G; Iurina, N P

    2009-01-01

    The effects of growth regulator of the new generation-melamine salt of bis(oxymethyl)phosphine acid (melafen)--on culture growth, pigment and protein content, and the induction of protective chloroplastic chaperone HSP70B in Chlamydomonas reinhardtii CW15 cells were studied. Melafen exhibited 10-30% growth inhibition at 10(-9)-10(-2)% concentration. At 10(-9)-10(-4)% of melafen electrophoretic concentration, the pattern of cellular proteins was similar to the control. The alterations in protein content of algae cells were detected only at 10(-2)% concentration. The content of chlorophyll and carotenoids in melafen-treated cells was 17-40% lower than in the control. Melafen at 10(-9)-109-2)% concentration inhibited HSP70B induction by 39-43% compared to untreated cells. The potential mechanism of melafen effect might involve its influence on nuclear gene expression.

  7. Prokaryotic expression of chicken interferon-γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress.

    Science.gov (United States)

    Sun, Jinhua; Chen, Yinglin; Qin, Feiyue; Guan, Xueting; Xu, Wei; Xu, Liangmei

    2017-06-01

    Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full-length chicken interferon-γ (ChIFN-γ) gene was amplified by RT-PCR. The full complementary DNA sequence of the ChIFN-γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX™HT b . The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN-γ was induced by isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti-His mAb. Moreover, ChIFN-γ was found to demonstrate anti-viral activity in vitro. To test the in vivo function of ChIFN-γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN-γ. Results demonstrated ChIFN-γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN-γ can reduce heat stress to some extent in vivo. © 2016 Japanese Society of Animal Science.

  8. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

    Science.gov (United States)

    2011-01-01

    Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells. PMID:21663599

  9. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

    Directory of Open Access Journals (Sweden)

    Lisowska Katarzyna Marta

    2011-06-01

    Full Text Available Abstract Background During functional studies on the rat stress-inducible Hspa1b (hsp70.1 gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA, Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. Results We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. Conclusions Our observations suggest that i some cationic liposomes may not be suitable for functional studies on hsp promoters, ii lipofection may cause unintended changes in global gene expression in the transfected cells.

  10. Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte.

    Science.gov (United States)

    Hiraoka, Nobuyuki; Arai, Yuji; Takahashi, Kenji A; Mazda, Osam; Kishida, Tsunao; Honjo, Kuniaki; Tsuchida, Shinji; Inoue, Hiroaki; Morino, Saori; Suico, Mary Ann; Kai, Hirofumi; Kubo, Toshikazu

    2013-06-01

    The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real-time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage. Copyright © 2013 Orthopaedic Research Society.

  11. Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

    International Nuclear Information System (INIS)

    Aloy, Marie-Therese; Hadchity, Elie; Bionda, Clara; Diaz-Latoud, Chantal; Claude, Line; Rousson, Robert; Arrigo, Andre-Patrick; Rodriguez-Lafrasse, Claire

    2008-01-01

    Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to γ-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors

  12. HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene.

    Science.gov (United States)

    Grably, Melanie R; Stanhill, Ariel; Tell, Osnat; Engelberg, David

    2002-04-01

    In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5' deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Deltamsn4Delta cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Delta cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Deltamsn2Deltamsn4Delta cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

  13. Dietary ascorbic acid modulates the expression profile of stress protein genes in hepatopancreas of adult Pacific abalone Haliotis discus hannai Ino.

    Science.gov (United States)

    Wu, Chenglong; Wang, Jia; Xu, Wei; Zhang, Wenbing; Mai, Kangsen

    2014-12-01

    This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg(-1) diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg(-1)) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg(-1) dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg(-1)). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg(-1)) as compared to those fed 70.3 mg kg(-1) dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg(-1)) reduced the oxidative stress

  14. Differential stress response of Saccharomyces hybrids revealed by monitoring Hsp104 aggregation and disaggregation.

    Science.gov (United States)

    Kempf, Claudia; Lengeler, Klaus; Wendland, Jürgen

    2017-07-01

    Proteotoxic stress may occur upon exposure of yeast cells to different stress conditions. The induction of stress response mechanisms is important for cells to adapt to changes in the environment and ensure survival. For example, during exposure to elevated temperatures the expression of heat shock proteins such as Hsp104 is induced in yeast. Hsp104 extracts misfolded proteins from aggregates to promote their refolding. We used an Hsp104-GFP reporter to analyze the stress profiles of Saccharomyces species hybrids. To this end a haploid S. cerevisiae strain, harboring a chromosomal HSP104-GFP under control of its endogenous promoter, was mated with stable haploids of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum. Stress response behaviors in these hybrids were followed over time by monitoring the appearance and dissolution of Hsp104-GFP foci upon heat shock. General stress tolerance of these hybrids was related to the growth rate detected during exposure to e.g. ethanol and oxidizing agents. We observed that hybrids were generally more resistant to high temperature and ethanol stress compared to their parental strains. Amongst the hybrids differential responses regarding the appearance of Hsp104-foci and the time required for dissolving these aggregates were observed. The S. cerevisiae/S. paradoxus hybrid, combining the two most closely related strains, performed best under these conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Differential effects of metal contamination on the transcript expression of immune- and stress-response genes in the Sydney Rock oyster, Saccostrea glomerata

    International Nuclear Information System (INIS)

    Taylor, Daisy A.; Thompson, Emma L.; Nair, Sham V.; Raftos, David A.

    2013-01-01

    Environmental contamination by metals is a serious threat to the biological sustainability of coastal ecosystems. Our current understanding of the potential biological effects of metals in these ecosystems is limited. This study tested the transcriptional expression of immune- and stress-response genes in Sydney Rock oysters (Saccostrea glomerata). Oysters were exposed to four metals (cadmium, copper, lead and zinc) commonly associated with anthropogenic pollution in coastal waterways. Seven target genes (superoxide dismutase, ferritin, ficolin, defensin, HSP70, HSP90 and metallothionein) were selected. Quantitative (real-time) PCR analyses of the transcript expression of these genes showed that each of the different metals elicited unique transcriptional profiles. Significant changes in transcription were found for 18 of the 28 combinations tested (4 metals × 7 genes). Of these, 16 reflected down-regulation of gene transcription. HSP90 was the only gene significantly up-regulated by metal contamination (cadmium and zinc only), while defensin expression was significantly down-regulated by exposure to all four metals. This inhibition could have a significant negative effect on the oyster immune system, promoting susceptibility to opportunistic infections and disease. -- Highlights: ► Oysters were exposed to Cd, Cu, Pb or Zn, all commonly associated with coastal pollution. ► qPCR identified significant down-regulation in stress- and immune-response genes in oysters exposed to these metals. ► qPCR showed that each of the different metals elicited unique transcriptional profiles. ► The genes identified have the potential to lead to increased disease susceptibility in oysters. -- qPCR identified significant down-regulation in stress- and immune-response genes in oysters exposed to metals, which could lead to increased disease susceptibility

  16. Differential expression of heat shock transcription factors and heat shock proteins after acute and chronic heat stress in laying chickens (Gallus gallus).

    Science.gov (United States)

    Xie, Jingjing; Tang, Li; Lu, Lin; Zhang, Liyang; Xi, Lin; Liu, Hsiao-Ching; Odle, Jack; Luo, Xugang

    2014-01-01

    Heat stress due to high environmental temperature negatively influences animal performances. To better understand the biological impact of heat stress, laying broiler breeder chickens were subjected either to acute (step-wisely increasing temperature from 21 to 35°C within 24 hours) or chronic (32°C for 8 weeks) high temperature exposure. High temperature challenges significantly elevated body temperature of experimental birds (Pshock transcription factors (HSFs) and heat shock proteins (HSPs) 70 and 90 were differently affected by acute and chronic treatment. Tissue-specific responses to thermal challenge were also found among heart, liver and muscle. In the heart, acute heat challenge affected lipid oxidation (P = 0.05) and gene expression of all 4 HSF gene expression was upregulated (Pstress increased protein oxidation, but HSFs and HSPs gene expression remained unaltered. Only tendencies to increase were observed in HSP 70 (P = 0.052) and 90 (P = 0.054) gene expression after acute heat stress. The differential expressions of HSF and HSP genes in different tissues of laying broiler breeder chickens suggested that anti-heat stress mechanisms might be provoked more profoundly in the heart, by which the muscle was least protected during heat stress. In addition to HSP, HSFs gene expression could be used as a marker during acute heat stress.

  17. A toxic imbalance of Hsp70s in Saccharomyces cerevisiae is caused by competition for cofactors.

    Science.gov (United States)

    Keefer, Kathryn M; True, Heather L

    2017-09-01

    Molecular chaperones are responsible for managing protein folding from translation through degradation. These crucial machines ensure that protein homeostasis is optimally maintained for cell health. However, 'too much of a good thing' can be deadly, and the excess of chaperones can be toxic under certain cellular conditions. For example, overexpression of Ssa1, a yeast Hsp70, is toxic to cells in folding-challenged states such as [PSI+]. We discovered that overexpression of the nucleotide exchange factor Sse1 can partially alleviate this toxicity. We further argue that the basis of the toxicity is related to the availability of Hsp70 cofactors, such as Hsp40 J-proteins and nucleotide exchange factors. Ultimately, our work informs future studies about functional chaperone balance and cautions against therapeutic chaperone modifications without a thorough examination of cofactor relationships. © 2017 John Wiley & Sons Ltd.

  18. Dietary supplementation of Zingiber officinale and Zingiber zerumbet to heat-stressed broiler chickens and its effect on heat shock protein 70 expression, blood parameters and body temperature.

    Science.gov (United States)

    Hasheimi, S R; Zulkifli, I; Somchit, M N; Zunita, Z; Loh, T C; Soleimani, A F; Tang, S C

    2013-08-01

    The present study was conducted to assess the effects of dietary supplementation of Zingiber officinale and Zingiber zerumbet and to heat-stressed broiler chickens on heat shock protein (HSP) 70 density, plasma corticosterone concentration (CORT), heterophil to lymphocyte ratio (HLR) and body temperature. Beginning from day 28, chicks were divided into five dietary groups: (i) basal diet (control), (ii) basal diet +1%Z. zerumbet powder (ZZ1%), (iii) basal diet +2%Z. zerumbet powder (ZZ2%), (iv) basal diet +1%Z. officinale powder (ZO1%) and (v) basal diet +2%Z. officinale powder (ZO2%). From day 35-42, heat stress was induced by exposing birds to 38±1°C and 80% RH for 2 h/day. Irrespective of diet, heat challenge elevated HSP70 expression, CORT and HLR on day 42. On day 42, following heat challenge, the ZZ1% birds showed lower body temperatures than those of control, ZO1% and ZO2%. Neither CORT nor HLR was significantly affected by diet. The ZO2% and ZZ2% diets enhanced HSP70 expression when compared to the control groups. We concluded that dietary supplementation of Z. officinale and Z. zerumbet powder may induce HSP70 reaction in broiler chickens exposed to heat stress. © 2012 Blackwell Verlag GmbH.

  19. Increased stocking density causes changes in expression of selected stress- and immune-related genes, humoral innate immune parameters and stress responses of rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Yarahmadi, Peyman; Miandare, Hamed Kolangi; Fayaz, Sahel; Caipang, Christopher Marlowe A

    2016-01-01

    The present study investigated the effects of various stocking densities on the health status (stress and immune responses) of rainbow trout (Onchorhynchus mykiss). Juvenile rainbow trout were acclimated, placed in circular tanks under stocking densities of 10, 40 and 80 kg m(-3) and reared for 30 days. The relative expression of genes involved in stress and immunity such as HSP70, LyzII, TNF-1α, IL-1β, IL-8 and IFN-γ1 in the head kidney was determined. Serum cortisol, ACTH, total antioxidant capacity, osmolality and lactate were measured after 30 days of culture at different stocking densities (D1:10 kg m(-3), D2: 40 kg m(-3) and D3: 80 kg m(-3)) as indices of stress responses. In addition, the effects of stocking densities on serum complement, bactericidal activity, agglutinating antibody titers, serum IgM, anti-protease activity, serum total protein and alkaline phosphatase of the fish were measured. HSP70 gene expression was significantly density-dependent upregulated in D2 and D3 densities compared to D1 (P < 0.05). Also, there was significant downregulation in expression of LyzII, TNF-1α, IL-1β, IL-8 and IFN-γ1 in fish reared at density of either D2 or D3 (P < 0.05). In terms of stress responses, serum ACTH, cortisol and lactate level showed significant density-dependent increase (P < 0.05) while serum osmolality and total antioxidant capacity showed significant decline (P < 0.05) in fish reared at higher densities (D2 and D3) compared to fish reared at lower density (D1) (P < 0.05). Concordant with the expression of the immune-related genes, the serum complement and bactericidal activity as well as specific antibody titer against Aeromonas hydrophila, IgM and anti-protease activity decreased along with elevation of stocking density from D1 to D3 (P < 0.05). However, different stocking densities had no significant effect on serum total protein level and alkaline phosphatase activity. These results suggested that elevation of stocking

  20. Endurance training alters YKL40, PERM1, and HSP70 skeletal muscle protein contents in men with type 2 diabetes mellitus.

    Science.gov (United States)

    Brinkmann, Christian; Kuckertz, Anika; Schiffer, Thorsten; Bloch, Wilhelm; Predel, Hans-Georg; Brixius, Klara

    2018-05-21

    The fight against type 2 diabetes mellitus (T2DM) is tremendously challenging. This pilot study investigates whether endurance training (3 times per week for 3 months, moderate intensity) can change the skeletal muscle protein contents of chitinase-3-like protein-1 (YKL40), peroxisome proliferator-activated receptor y coactivator-1 and estrogen-related receptor-induced regulator in muscle-1 (PERM1) and heat-shock protein-70 (HSP70), which have been discussed as novel therapeutically relevant targets. Muscle biopsies were obtained from overweight/obese men with T2DM (n = 7, years = 63 ± 9) at T1 (6 weeks pre-training), T2 (1 week pre-training) and T3 (3 to 4 days post-training). The protein levels of YKL40, PERM1, and HSP70 were determined by immunohistochemistry. YKL40, PERM1, and HSP70 were significantly upregulated following endurance training (T2-T3: +103%, +61%, +89%, p = 0.012, p = 0.010, p = 0.028). There was a fiber type-specific distribution of HSP70 with increased protein contents in type I fibers. A significant change in the fiber type distribution with an increase in type I fibers and a decrease in type II fibers was observed post-training. There were no significant differences for YKL40, PERM1, HSP70, or the fiber type distribution between T1 and T2. The training-induced upregulation of YKL40, PERM1, and HSP70 could help manage the diabetic disease and reduce its complications.

  1. Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats

    Science.gov (United States)

    Choudhary, Arbind Kumar; Devi, Rathinasamy Sheela

    2016-01-01

    Abstract Aspartame, a “first generation sweetener”, is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake (ADI) value of aspartame to be 50 mg/kg·day, while the JECFA (Joint FAO/WHO Expert Committee on Food Additives) has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/(kg·day)] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period (90-days) has any effect on the expression of hsp70, bcl-2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes. PMID:27845306

  2. Pharmacophore modeling, virtual screening and molecular docking of ATPase inhibitors of HSP70.

    Science.gov (United States)

    Sangeetha, K; Sasikala, R P; Meena, K S

    2017-10-01

    Heat shock protein 70 is an effective anticancer target as it influences many signaling pathways. Hence the study investigated the important pharmacophore feature required for ATPase inhibitors of HSP70 by generating a ligand based pharmacophore model followed by virtual based screening and subsequent validation by molecular docking in Discovery studio V4.0. The most extrapolative pharmacophore model (hypotheses 8) consisted of four hydrogen bond acceptors. Further validation by external test set prediction identified 200 hits from Mini Maybridge, Drug Diverse, SCPDB compounds and Phytochemicals. Consequently, the screened compounds were refined by rule of five, ADMET and molecular docking to retain the best competitive hits. Finally Phytochemical compounds Muricatetrocin B, Diacetylphiladelphicalactone C, Eleutheroside B and 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]amino}phenyl)- 4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic acid were obtained as leads to inhibit the ATPase activity of HSP70 in our findings and thus can be proposed for further in vitro and in vivo evaluation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Differential responses of the coral host and their algal symbiont to thermal stress.

    Directory of Open Access Journals (Sweden)

    William Leggat

    Full Text Available The success of any symbiosis under stress conditions is dependent upon the responses of both partners to that stress. The coral symbiosis is particularly susceptible to small increases of temperature above the long term summer maxima, which leads to the phenomenon known as coral bleaching, where the intracellular dinoflagellate symbionts are expelled. Here we for the first time used quantitative PCR to simultaneously examine the gene expression response of orthologs of the coral Acropora aspera and their dinoflagellate symbiont Symbiodinium. During an experimental bleaching event significant up-regulation of genes involved in stress response (HSP90 and HSP70 and carbon metabolism (glyceraldehyde-3-phosphate dehydrogenase, α-ketoglutarate dehydrogenase, glycogen synthase and glycogen phosphorylase from the coral host were observed. In contrast in the symbiont, HSP90 expression decreased, while HSP70 levels were increased on only one day, and only the α-ketoglutarate dehydrogenase expression levels were found to increase. In addition the changes seen in expression patterns of the coral host were much larger, up to 10.5 fold, compared to the symbiont response, which in all cases was less than 2-fold. This targeted study of the expression of key metabolic and stress genes demonstrates that the response of the coral and their symbiont vary significantly, also a response in the host transcriptome was observed prior to what has previously been thought to be the temperatures at which thermal stress events occur.

  4. Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment.

    Science.gov (United States)

    Yang, Guiyan; Wang, Yucheng; Zhang, Kaimin; Gao, Caiqiu

    2014-03-01

    Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1-9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.

  5. Phloroglucinol-Mediated Hsp70 Production in Crustaceans: Protection against Vibrio parahaemolyticus in Artemia franciscana and Macrobrachium rosenbergii

    Science.gov (United States)

    Kumar, Vikash; Baruah, Kartik; Nguyen, Dung Viet; Smagghe, Guy; Vossen, Els; Bossier, Peter

    2018-01-01

    The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND) in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30 µM), protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70) production (twofolds or more) as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30 µM) and Macrobrachium larvae (5 µM) with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.

  6. Phloroglucinol-Mediated Hsp70 Production in Crustaceans: Protection against Vibrio parahaemolyticus in Artemia franciscana and Macrobrachium rosenbergii

    Directory of Open Access Journals (Sweden)

    Vikash Kumar

    2018-05-01

    Full Text Available The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30 µM, protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70 production (twofolds or more as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30 µM and Macrobrachium larvae (5 µM with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.

  7. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  8. Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis).

    Science.gov (United States)

    Kapila, Neha; Sharma, Ankita; Kishore, Amit; Sodhi, Monika; Tripathi, Pawan K; Mohanty, Ashok K; Mukesh, Manishi

    2016-01-01

    The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative

  9. Induction of stress- and immune-associated genes in the Indian meal moth Plodia interpunctella against envenomation by the ectoparasitoid Bracon hebetor.

    Science.gov (United States)

    Shafeeq, Tahir; UlAbdin, Zain; Lee, Kyeong-Yeoll

    2017-10-01

    Envenomation is an important process in parasitism by parasitic wasps; it suppresses the immune and development of host insects. However, the molecular mechanisms of host responses to envenomation are not yet clear. This study aimed to determine the transcription-level responses of the Indian meal moth Plodia interpunctella against envenomation of the ectoparasitoid Bracon hebetor. Quantitative real-time reverse-transcription PCR was used to determine the transcriptional changes of 13 selected genes, which are associated with development, metabolism, stress, or immunity, in the feeding and wandering fifth instar larvae over a 4-day period after envenomation. The effects of envenomation on the feeding-stage larvae were compared with those of starvation in the transcriptional levels of the 13 genes. Most selected genes were altered in their expression by either envenomation or starvation. In particular, a heat shock protein, hsp70, was highly upregulated in envenomated larvae in both the feeding and wandering stages as well as in starved larvae. Further, some genes were upregulated by envenomation in a stage-specific manner. For example, hsp25 was upregulated after envenomation in the feeding larvae, but hsp90 and an immune-associated gene, hemolin, were upregulated in the wandering larvae. However, both envenomation and starvation resulted in the downregulation of genes associated with development and metabolism. Taken together, P. interpunctella upregulated stress- and immune-responsive genes, but downregulated genes associated with development and metabolism after envenomation. This study provides important information for understanding the molecular mechanisms of host responses to parasitism. © 2017 Wiley Periodicals, Inc.

  10. Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

    Science.gov (United States)

    Hazra, Joyita; Mukherjee, Pooja; Ali, Asif; Poddar, Soumita; Pal, Mahadeb

    2017-01-01

    An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1.

  11. In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin

    Science.gov (United States)

    Wilmink, Gerald J.; Opalenik, Susan R.; Beckham, Joshua T.; Mackanos, Mark A.; Nanney, Lillian B.; Contag, Christopher H.; Davidson, Jeffrey M.; Jansen, E. Duco

    2013-01-01

    Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 μm), protein (amide-II band, 6.45 μm), and both water and protein (amide-I band, 6.10 μm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 μm have approximately 4× higher hsp70 expression than 6.10 μm. Histology shows that under comparable fluences, tissue injury at the 2.94-μm wavelength was 2× and 3× deeper than 6.45 and 6.10 μm, respectively. The 6.10-μm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-μm wavelength is a superior wavelength for laser ablation of skin. PMID:19021444

  12. Heat Shock Protein Genes Undergo Dynamic Alteration in Their Three-Dimensional Structure and Genome Organization in Response to Thermal Stress.

    Science.gov (United States)

    Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

    2017-12-15

    Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein ( HSP ) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci. Copyright © 2017 American Society for Microbiology.

  13. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  14. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

    Science.gov (United States)

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  15. Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

    Science.gov (United States)

    Jespersen, David; Belanger, Faith C; Huang, Bingru

    2017-01-01

    Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

  16. Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

    Directory of Open Access Journals (Sweden)

    David Jespersen

    Full Text Available Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L. x creeping bentgrass (Agrostis stolonifera L. hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease, antioxidant defense (catalase and glutathione-S-transferase, energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, cell expansion (expansin, and stress protection (heat shock proteins HSP26, HSP70, and HSP101. Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

  17. ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-01-01

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

  18. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

    Science.gov (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

    2010-02-12

    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  19. Wheat chloroplast targeted sHSP26 promoter confers heat and abiotic stress inducible expression in transgenic Arabidopsis Plants.

    Directory of Open Access Journals (Sweden)

    Neetika Khurana

    Full Text Available The small heat shock proteins (sHSPs have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs in the promoter of sHSP26 was performed. Moreover, the importance of 5' untranslated region (UTR has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress.

  20. Effect of thermal stress on HSP90 expression of Bali cattle in Barru district, South Sulawesi

    Science.gov (United States)

    Aritonang, S. B.; Yuniati, R.; Abinawanto, Imron, M.; Bowolaksono, A.

    2017-07-01

    Heat shock protein 90-kDa is induced stress protein that expressed in response to stress and play crucial roles in environmental stress tolerance and adaptation. This study aimed to determine effect of environmental heat stress on the HSP90 expression of Bali cattle. Heat stress was measured by temperature humidity index in the morning and evening across 5-days on August 2016. The blood samples of Bali cattle were taken from venous jungularis. HSP90 was derived from RNA isolation of whole blood then was followed reverse transcription two steps. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to analyze the transcript variants of HSP90, followed by comparative ΔΔCt to determine HSP90 expression. The results of temperature and humidity index (THI) measurement indicated THI on afternoon was higher than in the morning. The difference in environmental conditions in the morning and afternoon effected changes on rectal temperature but neither did on Hsp90 expression.

  1. Induction of heat shock proteins (hsp70) in the zebra mussel (Dreissena polymorpha) following exposure to platinum group metals (platinum, palladium and rhodium): Comparison with lead and cadmium exposures

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Christoph [Zoologisches Institut I-Oekologie, Universitaet Karlsruhe, Geb. 07.01, Kornblumenstrasse 13, D-76131 Karlsruhe (Germany); Zimmermann, Sonja [Zoologisches Institut I-Oekologie, Universitaet Karlsruhe, Geb. 07.01, Kornblumenstrasse 13, D-76131 Karlsruhe (Germany); Sures, Bernd [Zoologisches Institut I-Oekologie, Universitaet Karlsruhe, Geb. 07.01, Kornblumenstrasse 13, D-76131 Karlsruhe (Germany)]. E-mail: dc11@rz.uni-karlsruhe.de

    2005-10-05

    An increasing number of papers concentrate on the availability and uptake of platinum group elements (PGE) by different organisms. These metals are discharged into the environment from different anthropogenic sources, such as automobile catalytic converters, hospitals and other medical institutions. As the effects of these precious metals on organisms remain unclear, the induction of heat shock proteins (hsp70) in zebra mussels (Dreissena polymorpha) following exposure to soluble salts of platinum, palladium and rhodium was compared with the hsp70 induction in mussels following exposure to cadmium and lead. Mussels were sampled weekly during a period of 10 weeks and analyzed for their metal concentration and their hsp70 level. Highest metal uptake was found for Cd, followed by Pt, Pb and Pd. Rh demonstrated the lowest uptake rate. A clear time-dependent increase of hsp70 levels occurred in all exposed mussels. Concentrations of hsp70 started to rise between days 18 and 25, except for the Pt-exposed group, where the initial increase was between days 25 and 32. All groups reached maximal hsp70 concentrations at day 39. Subsequently, hsp70 levels decreased to initial levels for the remaining exposure period. Threshold metal levels for the hsp70 induction varied among the metals and increased in the order: Rh < Pd {<=} Pb < Pt < Cd. Highest hsp70 values were found for mussels exposed to Pd, with a 25-fold higher level than in the controls, followed by Pt- and Rh-exposed mussels, which showed a 19-fold increase. The hsp70 levels of the mussels exposed to Cd and Pb were much lower, showing 6- and 12-fold higher values than the control, respectively. The clear induction of hsp70 due to exposure to Pt, Pd and Rh gives evidence for strong cellular effects of these metals, especially, when compared with lead and cadmium. Among the metals tested, Pd seems to have the highest potential as inducer for hsp70 production due to its low threshold level in combination with the

  2. Induction of heat shock proteins (hsp70) in the zebra mussel (Dreissena polymorpha) following exposure to platinum group metals (platinum, palladium and rhodium): Comparison with lead and cadmium exposures

    International Nuclear Information System (INIS)

    Singer, Christoph; Zimmermann, Sonja; Sures, Bernd

    2005-01-01

    An increasing number of papers concentrate on the availability and uptake of platinum group elements (PGE) by different organisms. These metals are discharged into the environment from different anthropogenic sources, such as automobile catalytic converters, hospitals and other medical institutions. As the effects of these precious metals on organisms remain unclear, the induction of heat shock proteins (hsp70) in zebra mussels (Dreissena polymorpha) following exposure to soluble salts of platinum, palladium and rhodium was compared with the hsp70 induction in mussels following exposure to cadmium and lead. Mussels were sampled weekly during a period of 10 weeks and analyzed for their metal concentration and their hsp70 level. Highest metal uptake was found for Cd, followed by Pt, Pb and Pd. Rh demonstrated the lowest uptake rate. A clear time-dependent increase of hsp70 levels occurred in all exposed mussels. Concentrations of hsp70 started to rise between days 18 and 25, except for the Pt-exposed group, where the initial increase was between days 25 and 32. All groups reached maximal hsp70 concentrations at day 39. Subsequently, hsp70 levels decreased to initial levels for the remaining exposure period. Threshold metal levels for the hsp70 induction varied among the metals and increased in the order: Rh < Pd ≤ Pb < Pt < Cd. Highest hsp70 values were found for mussels exposed to Pd, with a 25-fold higher level than in the controls, followed by Pt- and Rh-exposed mussels, which showed a 19-fold increase. The hsp70 levels of the mussels exposed to Cd and Pb were much lower, showing 6- and 12-fold higher values than the control, respectively. The clear induction of hsp70 due to exposure to Pt, Pd and Rh gives evidence for strong cellular effects of these metals, especially, when compared with lead and cadmium. Among the metals tested, Pd seems to have the highest potential as inducer for hsp70 production due to its low threshold level in combination with the

  3. High-Fat Diet Induces Oxidative Stress and MPK2 and HSP83 Gene Expression in Drosophila melanogaster.

    Science.gov (United States)

    Trindade de Paula, Mariane; Poetini Silva, Márcia Rósula; Machado Araujo, Stífani; Cardoso Bortolotto, Vandreza; Barreto Meichtry, Luana; Zemolin, Ana Paula Pegoraro; Wallau, Gabriel L; Jesse, Cristiano Ricardo; Franco, Jeferson Luís; Posser, Thaís; Prigol, Marina

    2016-01-01

    The consumption of a high-fat diet (HFD) causes alteration in normal metabolism affecting lifespan of flies; however molecular mechanism associated with this damage in flies is not well known. This study evaluates the effects of ingestion of a diet supplemented with 10% and 20% of coconut oil, which is rich in saturated fatty acids, on oxidative stress and cells stress signaling pathways. After exposure to the diet for seven days, cellular and mitochondrial viability, lipid peroxidation and antioxidant enzymes SOD and CAT activity, and mRNA expression of antioxidant enzymes HSP83 and MPK2 were analyzed. To confirm the damage effect of diet on flies, survival and lifespan were investigated. The results revealed that the HFD augmented the rate of lipid peroxidation and SOD and CAT activity and induced a higher expression of HSP83 and MPK2 mRNA. In parallel, levels of enzymes involved in lipid metabolism (ACSL1 and ACeCS1) were increased. Our data demonstrate that association among metabolic changes, oxidative stress, and protein signalization might be involved in shortening the lifespan of flies fed with a HFD.

  4. High-Fat Diet Induces Oxidative Stress and MPK2 and HSP83 Gene Expression in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Mariane Trindade de Paula

    2016-01-01

    Full Text Available The consumption of a high-fat diet (HFD causes alteration in normal metabolism affecting lifespan of flies; however molecular mechanism associated with this damage in flies is not well known. This study evaluates the effects of ingestion of a diet supplemented with 10% and 20% of coconut oil, which is rich in saturated fatty acids, on oxidative stress and cells stress signaling pathways. After exposure to the diet for seven days, cellular and mitochondrial viability, lipid peroxidation and antioxidant enzymes SOD and CAT activity, and mRNA expression of antioxidant enzymes HSP83 and MPK2 were analyzed. To confirm the damage effect of diet on flies, survival and lifespan were investigated. The results revealed that the HFD augmented the rate of lipid peroxidation and SOD and CAT activity and induced a higher expression of HSP83 and MPK2 mRNA. In parallel, levels of enzymes involved in lipid metabolism (ACSL1 and ACeCS1 were increased. Our data demonstrate that association among metabolic changes, oxidative stress, and protein signalization might be involved in shortening the lifespan of flies fed with a HFD.

  5. A comparative evaluation of crowding stress on muscle HSP90 and myostatin expression in salmonids

    Science.gov (United States)

    Galt, Nicholas J.; Froehlich, Jacob Michael; McCormick, Stephen; Biga, Peggy R.

    2018-01-01

    Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin, and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48 h crowding stress. Crowding stress resulted in a decrease of myostatin-1ain brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1awas upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.

  6. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Zampieri

    2016-02-01

    Full Text Available Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.Exploring the High Resolution Melting (HRM dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR targeting heat-shock protein 70 coding gene (hsp70 revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania infantum chagasi, L. (L. amazonensis, L. (L. mexicana, L. (Viannia lainsoni, L. (V. braziliensis, L. (V. guyanensis, L. (V. naiffi and L. (V. shawi, and three species found in Eurasia and Africa, including L. (L. tropica, L. (L. donovani and L. (L. major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  7. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

    Science.gov (United States)

    Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

    2016-02-01

    Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  8. Effects of heated hydrotherapy on muscle HSP70 and glucose metabolism in old and young vervet monkeys.

    Science.gov (United States)

    Kavanagh, Kylie; Davis, Ashely T; Jenkins, Kurt A; Flynn, D Mickey

    2016-07-01

    Increasing heat shock protein 70 (HSP70) in aged and/or insulin-resistant animal models confers benefits to healthspan and lifespan. Heat application to increase core temperature induces HSPs in metabolically important tissues, and preliminary human and animal data suggest that heated hydrotherapy is an effective method to achieve increased HSPs. However, safety concerns exist, particularly in geriatric medicine where organ and cardiovascular disease commonly will preexist. We evaluated young vervet monkeys compared to old, insulin-resistant vervet monkeys (Chlorocebus aethiops sabaeus) in their core temperatures, glucose tolerance, muscle HSP70 level, and selected safety biomarkers after 10 sessions of hot water immersions administered twice weekly. Hot water immersion robustly induced the heat shock response in muscles. We observed that heat-treated old and young monkeys have significantly higher muscle HSP70 than control monkeys and treatment was without significant adverse effects on organ or cardiovascular health. Heat therapy improved pancreatic responses to glucose challenge and tended to normalize glucose excursions. A trend for worsened blood pressure and glucose values in the control monkeys and improved values in heat-treated monkeys were seen to support further investigation into the safety and efficacy of this intervention for metabolic syndrome or diabetes in young or old persons unable to exercise.

  9. Mechanisms of stress-induced cellular HSP72 release: implications for exercise-induced increases in extracellular HSP72.

    Science.gov (United States)

    Lancaster, Graeme I; Febbraio, Mark A

    2005-01-01

    The heat shock proteins are a family of highly conserved proteins with critical roles in maintaining cellular homeostasis and in protecting the cell from stressful conditions. While the critical intracellular roles of heat shock proteins are undisputed, evidence suggests that the cell possess the necessary machinery to actively secrete specific heat shock proteins in response to cellular stress. In this review, we firstly discuss the evidence that physical exercise induces the release of heat shock protein 72 from specific tissues in humans. Importantly, it appears as though this release is the result of an active secretory process, as opposed to non-specific processes such as cell lysis. Next we discuss recent in vitro evidence that has identified a mechanistic basis for the observation that cellular stress induces the release of a specific subset of heat shock proteins. Importantly, while the classical protein secretory pathway does not seem to be involved in the stress-induced release of HSP72, we discuss the evidence that lipid-rafts and exosomes are important mediators of the stress-induced release of HSP72.

  10. Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Science.gov (United States)

    Dores-Silva, Paulo R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2015-01-01

    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

  11. Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

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    Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2017-06-06

    Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

  12. The role of heat shock protein 70 in mediating age-dependent mortality in sepsis.

    Science.gov (United States)

    McConnell, Kevin W; Fox, Amy C; Clark, Andrew T; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A; Farris, Alton B; Buchman, Timothy G; Hunt, Clayton R; Coopersmith, Craig M

    2011-03-15

    Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70(-/-) and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70(-/-) and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70(-/-) mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70(-/-) mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70(-/-) mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

  13. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

    DEFF Research Database (Denmark)

    Schmitz-Abe, Klaus; Ciesielski, Szymon J.; Schmidt, Paul J.

    2015-01-01

    The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q...

  14. Sensing the Heat Stress by Mammalian Cells

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    Cates Jordan

    2011-08-01

    Full Text Available Abstract Background The heat-shock response network controls the adaptation and survival of the cell against environmental stress. This network is highly conserved and is connected with many other signaling pathways. A key element of the heat-shock network is the heat-shock transcription factor-1 (HSF, which is transiently activated by elevated temperatures. HSF translocates to the nucleus upon elevated temperatures, forming homotrimeric complexes. The HSF homotrimers bind to the heat shock element on the DNA and control the expression of the hsp70 gene. The Hsp70 proteins protect cells from thermal stress. Thermal stress causes the unfolding of proteins, perturbing thus the pathways under their control. By binding to these proteins, Hsp70 allows them to refold and prevents their aggregation. The modulation of the activity of the hsp70-promoter by the intensity of the input stress is thus critical for cell's survival. The promoter activity starts from a basal level and rapidly increases once the stress is applied, reaches a maximum level and attenuates slowely back to the basal level. This phenomenon is the hallmark of many experimental studies and of all computational network analysis. Results The molecular construct used as a measure of the response to thermal stress is a Hsp70-GFP fusion gene transfected in Chinese hamster ovary (CHO cells. The time profile of the GFP protein depends on the transient activity, Transient(t, of the heat shock system. The function Transient(t depends on hsp70 promoter activity, transcriptional regulation and the translation initiation effects elicited by the heat stress. The GFP time profile is recorded using flow cytometry measurements, a technique that allows a quantitative measurement of the fluorescence of a large number of cells (104. The GFP responses to one and two heat shocks were measured for 261 conditions of different temperatures and durations. We found that: (i the response of the cell to two

  15. Alteration of Transcripts of Stress-Protective Genes and Transcriptional Factors by γ-Aminobutyric Acid (GABA Associated with Improved Heat and Drought Tolerance in Creeping Bentgrass (Agrostis stolonifera

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    Zhou Li

    2018-05-01

    Full Text Available Gamma-aminobutyric acid (GABA may play a positive role in regulating plant tolerance to drought or heat stress. The objectives of this study were to investigate the physiological effects of GABA on tolerance of creeping bentgrass (Agrostis stolonifera to heat and drought stress and to determine whether enhanced heat and drought tolerance due to GABA treatment was associated with the up-regulation of selected genes and transcriptional factors involved in stress protection. Creeping bentgrass (cultivar “Penncross” plants were treated with 0.5 mM GABA or water (untreated control as a foliar spray and were subsequently exposed to heat stress (35/30 °C, day/night, drought stress by withholding irrigation, or non-stress conditions in controlled-environment growth chambers. Exogenous application of GABA significantly improved plant tolerance to heat and drought stress, as reflected by increased leaf water content, cell membrane stability, and chlorophyll content. The analysis of gene transcript level revealed that exogenous GABA up-regulated the expression of ABF3, POD, APX, HSP90, DHN3, and MT1 during heat stress and the expression of CDPK26, MAPK1, ABF3, WRKY75, MYB13, HSP70, MT1, 14-3-3, and genes (SOD, CAT, POD, APX, MDHAR, DHAR, and GR encoding antioxidant enzymes during drought stress. The up-regulation of the aforementioned stress-protective genes and transcriptional factors could contribute to improved heat and drought tolerance in creeping bentgrass.

  16. Alteration of Transcripts of Stress-Protective Genes and Transcriptional Factors by γ-Aminobutyric Acid (GABA) Associated with Improved Heat and Drought Tolerance in Creeping Bentgrass (Agrostis stolonifera).

    Science.gov (United States)

    Li, Zhou; Peng, Yan; Huang, Bingru

    2018-05-31

    Gamma-aminobutyric acid (GABA) may play a positive role in regulating plant tolerance to drought or heat stress. The objectives of this study were to investigate the physiological effects of GABA on tolerance of creeping bentgrass ( Agrostis stolonifera ) to heat and drought stress and to determine whether enhanced heat and drought tolerance due to GABA treatment was associated with the up-regulation of selected genes and transcriptional factors involved in stress protection. Creeping bentgrass (cultivar "Penncross") plants were treated with 0.5 mM GABA or water (untreated control) as a foliar spray and were subsequently exposed to heat stress (35/30 °C, day/night), drought stress by withholding irrigation, or non-stress conditions in controlled-environment growth chambers. Exogenous application of GABA significantly improved plant tolerance to heat and drought stress, as reflected by increased leaf water content, cell membrane stability, and chlorophyll content. The analysis of gene transcript level revealed that exogenous GABA up-regulated the expression of ABF3 , POD , APX , HSP90 , DHN3 , and MT1 during heat stress and the expression of CDPK26 , MAPK1 , ABF3 , WRKY75 , MYB13 , HSP70 , MT1 , 14-3-3 , and genes ( SOD , CAT , POD , APX , MDHAR , DHAR , and GR ) encoding antioxidant enzymes during drought stress. The up-regulation of the aforementioned stress-protective genes and transcriptional factors could contribute to improved heat and drought tolerance in creeping bentgrass.

  17. Encoded novel forms of HSP70 or a cytolytic protein increase DNA vaccine potency.

    Science.gov (United States)

    Garrod, Tamsin; Grubor-Bauk, Branka; Yu, Stanley; Gargett, Tessa; Gowans, Eric J

    2014-01-01

    In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen.

  18. Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

    Science.gov (United States)

    Alvares, K; Carrillo, A; Yuan, P M; Kawano, H; Morimoto, R I; Reddy, J K

    1990-01-01

    Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site. Images PMID:2371272

  19. The interactive association between heat shock factor 1 and heat shock proteins in primary myocardial cells subjected to heat stress.

    Science.gov (United States)

    Tang, Shu; Chen, Hongbo; Cheng, Yanfen; Nasir, Mohammad Abdel; Kemper, Nicole; Bao, Endong

    2016-01-01

    Heat shock factor 1 (HSF1) is a heat shock transcription factor that rapidly induces heat shock gene transcription following thermal stress. In this study, we subjected primary neonatal rat myocardial cells to heat stress in vitro to create a model system for investigating the trends in expression and association between various heat shock proteins (HSPs) and HSF1 under adverse environmental conditions. After the cells were subjected to heat stress at 42˚C for different periods of time, HSP and HSF1 mRNA and protein levels were detected by qPCR and western blot analysis in the heat-stressed cells. The HSF1 expression levels significantly increased in the cells following 120 min of exposure to heat stess compared to the levels observed at the beginning of heat stress exposure. HSP90 followed a similar trend in expression to HSF1, whereas HSP70 followed an opposite trend. However, no significant changes were observed in the crystallin, alpha B (CRYAB, also known as HSP beta-5) expression levels during the 480‑min period of exposure to heat stress. The interaction between the HSPs and HSF1 was analyzed by STRING 9.1, and it was found that HSF1 interacted with HSP90 and HSP70, and that it did not play a role in regulating CRYAB expression. Based on our findings, HSP70 may suppress HSF1 in rat myocardial cells under conditions of heat stress. Furthermore, our data demonstrate that HSF1 is not the key factor for all HSPs, and this was particularly the case for CRYAB.

  20. A fraction from Petiveria alliacea induces apoptosis via a mitochondria-dependent pathway and regulates HSP70 expression

    Directory of Open Access Journals (Sweden)

    Susana Fiorentino

    2009-12-01

    Full Text Available To evaluate the biological activity of Petiveria alliacea extracts on tumoral cells in vitro. Materials and methods. P.alliaceafractions prepared by a bioguided purification protocol were characterized by their biological activities on two human tumoral cell lines.Morphological changes, cell viability, mitochondrial membrane depolarization, nuclear staining and activity on HSP70 were analyzed.Results. The present study demonstrates that P.alliacea fractions can induce apoptosis in a mitochondria-dependent pathway and downregulate HSP70 expression in vitro. The possible compounds present in P.alliacea responsible for the described biological activities wereidentified by dereplication methods. Conclusion. The present study provides new knowledge on the antitumoral properties of the plantspecies P. alliacea described in several ethnobotanical studies.

  1. In situ mapping of the hsp70 locus in seven species of the willistoni group of Drosophila

    International Nuclear Information System (INIS)

    Bonorino, C.B.C.; Valente, V.L.S.; Pereira, M.; Alonso, C.E.V.; Abdelhay, E.

    1993-01-01

    The hsp70 locus was mapped by in situ hybridization of a biotinylated probe (p PW 229) to the polythene chromosomes of seven species of the willistoni group of Drosophila. In all of them, the probe hybridized mainly at a single site of the third chromosome, corresponding in each case to a heat-induced puff site. Southern blot analysis comparing the Eco RI digested DNA of the willistoni species with D. melanogaster revealed that a large segment of DNA homologous to the hsp 70 probe detected in D. melanogaster does not exist in the willistoni group. These results suggested that this locus is not duplicated in the willistoni group as it is in the melanogaster. (author)

  2. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Ute Hofmann

    2014-06-01

    Full Text Available In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells expressing green fluorescent protein (GFP under the control of the stress-inducible HSP70B’ promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B’. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B’ gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B’ promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  3. Oxygen sensors and energy sensors act synergistically to achieve a graded alteration in gene expression: consequences for assessing the level of neuroprotection in response to stressors.

    Science.gov (United States)

    Renshaw, Gillian M C; Warburton, Joshua; Girjes, Adeeb

    2004-01-01

    Changes in gene expression are associated with switching to an autoprotected phenotype in response to environmental and physiological stress. Ubiquitous molecular chaperones from the heat shock protein (HSP) superfamily confer neuronal protection that can be blocked by antibodies. Recent research has focused on the interactions between the molecular sensors that affect the increased expression of neuroprotective HSPs above constitutive levels. An examination of the conditions under which the expression of heat shock protein 70 (Hsp70) was up regulated in a hypoxia and anoxia tolerant tropical species, the epaulette shark (Hemiscyllium ocellatum), revealed that up-regulation was dependent on exceeding a stimulus threshold for an oxidative stressor. While hypoxic-preconditioning confers neuroprotective changes, there was no increase in the level of Hsp70 indicating that its increased expression was not associated with achieving a neuroprotected state in response to hypoxia in the epaulette shark. Conversely, there was a significant increase in Hsp70 in response to anoxic-preconditioning, highlighting the presence of a stimulus threshold barrier and raising the possibility that, in this species, Hsp70 contributes to the neuroprotective response to extreme crises, such as oxidative stress. Interestingly, there was a synergistic effect of coincident stressors on Hsp70 expression, which was revealed when metabolic stress was superimposed upon oxidative stress. Brain energy charge was significantly lower when adenosine receptor blockade, provided by treatment with aminophylline, was present prior to the final anoxic episode, under these circumstances, the level of Hsp70 induced was significantly higher than in the pair-matched saline treated controls. An understanding of the molecular and metabolic basis for neuroprotective switches, which result in an up-regulation of neuroprotective Hsp70 expression in the brain, is needed so that intervention strategies can be devised

  4. Noradrenaline increases the expression and release of Hsp72 by human neutrophils.

    Science.gov (United States)

    Giraldo, E; Multhoff, G; Ortega, E

    2010-05-01

    The blood concentration of extracellular 72kDa heat shock protein (eHsp72) increases under conditions of stress, including intense exercise. However, the signal(s), source(s), and secretory pathways in its release into the bloodstream have yet to be clarified. The aim of the present study was to evaluate the role of noradrenaline (NA) as a stress signal on the expression and release of Hsp72 by circulating neutrophils (as a source), all within a context of the immunophysiological regulation during exercise-induced stress in sedentary and healthy young (21-26years) women. The expression of Hsp72 on the surface of isolated neutrophils was determined by flow cytometry, and its release by cultured isolated neutrophils was determined by ELISA. Incubation with cmHsp70-FITC showed that neutrophils express Hsp72 on their surface under basal conditions. In addition, cultured isolated neutrophils (37 degrees C and 5% CO(2)) also released Hsp72 under basal conditions, with this release increasing from 10min to 24h in the absence of cell damage. NA at 10(-9)-10(-5)M doubled the percentage of neutrophils expressing Hsp72 after 60min and 24h incubation. NA also stimulated (by about 20%) the release of Hsp72 after 10min of incubation. (1) Hsp72 is expressed on the surface of isolated neutrophils under basal conditions, and this expression is augmented by NA. (2) Isolated neutrophils can also release Hsp72 under cultured basal conditions in the absence of cell death, and NA can increase this release. These results may contribute to confirming the hypothesis that NA can act as a "stress signal" for the increased eHsp72 in the context of exercise stress, with a role for neutrophils as a source for the expression and, to a lesser degree, the release of Hsp72 after activation by NA. Copyright 2010 Elsevier Inc. All rights reserved.

  5. Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in Candida albicans

    Science.gov (United States)

    Mayer, François L.; Wilson, Duncan; Jacobsen, Ilse D.; Miramón, Pedro; Slesiona, Silvia; Bohovych, Iryna M.; Brown, Alistair J. P.; Hube, Bernhard

    2012-01-01

    Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first

  6. The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

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    Nadja Kettern

    Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

  7. TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat (Triticum aestivum L.) including previously unknown Hsf targets.

    Science.gov (United States)

    Xue, Gang-Ping; Drenth, Janneke; McIntyre, C Lynne

    2015-02-01

    Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. Vinclozolin alters the expression of hormonal and stress genes in the midge Chironomus riparius.

    Science.gov (United States)

    Aquilino, Mónica; Sánchez-Argüello, Paloma; Martínez-Guitarte, José-Luis

    2016-05-01

    Vinclozolin is a fungicide used in agriculture that can reach aquatic ecosystems and affect the organisms living there. Its effects have been intensively studied in vertebrates, where it acts as an antiandrogen, but there is a lack of information about its mechanistic effects on invertebrates. In this work, we analyzed the response of genes related to the endocrine system, the stress response, and the detoxification mechanisms of Chironomus riparius fourth instar larvae after 24h and 48h exposures to 20 (69.9nM), 200 (699nM), and 2000μg/L (6.99μM) of Vinclozolin. Survival analysis showed that this compound has low toxicity, as it was not lethal for this organism at the concentrations used. However, this fungicide was shown to modify the transcriptional activity of the ecdysone response pathway genes EcR, E74, and Kr-h1 by increasing their mRNA levels. While no changes were observed in disembodied, a gene related with the ecdysone synthesis metabolic pathway, Cyp18A1, which is involved in the inactivation of the active form of ecdysone, was upregulated. Additionally, the expression of two genes related to other hormones, FOXO and MAPR, did not show any changes when Vinclozolin was present. The analysis of stress response genes showed significant changes in the mRNA levels of Hsp70, Hsp24, and Gp93, indicating that Vinclozolin activates the cellular stress mechanisms. Finally, the expressions of the genes Cyp4G and GstD3, which encode enzymes involved in phase I and phase II detoxification, respectively, were analyzed. It was found that their mRNA levels were altered by Vinclozolin, suggesting their involvement in the degradation of this compound. For the first time, these results show evidence that Vinclozolin can modulate gene expression, leading to possible significant endocrine alterations of the insect endocrine system. These results also offer new clues about the mode of action of this compound in invertebrates. Copyright © 2016 Elsevier B.V. All rights

  9. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

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    Zhengyi Liu

    2014-01-01

    Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

  10. Caracterización in silico de las proteínas del choque térmico Hsp70 y Hsp90 deBemisia tabaci (Hemiptera: Aleyrodidae y su posible actividad adaptativa

    Directory of Open Access Journals (Sweden)

    Eneida Torres Cabra

    2014-06-01

    Full Text Available La mosca blanca, Bemisia tabaci (Hemiptera: Aleyrodidae es una de las plagas más destructivas e invasivas en el mundo, ataca una gran cantidad de cultivos. Se adapta fácilmente a plantas hospederas y a nuevas regiones geográficas, lo que sugiere el desarrollo de mecanismos de control a daños producidos por factores estresantes. Las proteínas Hsp se expresanen los organismos como mecanismo de defensa, actúan como chaperonas en el correcto ensamblaje de las proteínas. En este estudio se realizó una caracterizaciónin silico de las proteínas Hsp70 y Hsp90 de B. tabaci, secuencias obtenidas de NCBI. La determinaciónde los perfiles de hidrofobicidad, polaridad, accesibilidady flexibilidad se obtuvieron con “ProScale” de ExPASy, el perfil de antigenicidad con JaMBW. La secuencia aminoacídica se analizó con GOR IV y SOPMA y la composición de aminoácidos con ProtParam. Para analizar el peso molecular, índice deinestabilidad, índice alifático y gradiente hidropático,con GRAVY. La estructura terciaria se obtuvo con HHpred, y ESyPred3D. Para validar las estructuras 3D se utilizó Procheck, What_check y errat. Hsp70 y Hsp90 de B. tabaci presentan valores bajos de hidrofobicidady altos de polaridad, flexibilidad y accesibilidad, características que le permiten a las proteínas extender su capacidad como chaperonas. La Hsp70tiene una estructura secundaria compuesta por 41-45% alfa hélices, 30-43% coil y menos del 6% en hoja plegada y la Hsp90 por 52 y 53% hélices, 26-34% coily 6% hoja plegada. Las Hsp juegan un rol importante en los insectos debido a su tamaño y corto ciclo de vida, pues la temperatura influye en su distribución y abundancia.

  11. Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

    International Nuclear Information System (INIS)

    Mao Li; Bryantsev, Anton L.; Chechenova, Maria B.; Shelden, Eric A.

    2005-01-01

    Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function

  12. Genome-wide analysis of rice ClpB/HSP100, ClpC and ClpD genes

    Directory of Open Access Journals (Sweden)

    Mittal Dheeraj

    2010-02-01

    Full Text Available Abstract Background ClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues. Results Nine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300, 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770 and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210. Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Δhsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene

  13. Heat shock protein Hsp90-2 expression in the Arabidopsis thaliana seedlings under clinorotation

    Science.gov (United States)

    Kozeko, Liudmyla

    Heat shock proteins 90 kDa (Hsp90) are abundant under normal conditions and induced by stress. This family is distinguished from other chaperones in that most of its substrates are signal transduction proteins. Previously, we determined some time-dependent increase in the Hsp90 level in pea seedlings in response to simulated microgravity that indicated a stress-reaction. However, expression of the individual members of the Hsp90 family have specific pattern. The purpose of this study was to investigate possible alterations in the gene expression pattern of cytosolic Hsp90-2 in Arabidopsis thaliana seedlings under 2D-clinorotation. To obtain detailed expression pattern of the HSP90-2 genes we used seeds that provides a resource of loss-of-function mutations gene expression patterns via translational fusions with the reporter gene, GUS (a line N 166718, NASC). There were two variants of the experiment: 1) seedlings grew under clinorotation for 10, 12, 14 d; 2) seedlings grew in the stationary conditions for 10 d followed by clinorotation for 3 h -at 22o C and 16h light cycle. The seedlings grown in the stationary conditions were used as a control. GUS staining showed that HSP90-2 expression was regulated during seedling development and affected by clinorotation in the heterozygous mutant plants. In the homozygous for the mutation plants, HSP90-2 expression was stable during seedling development and not affected by clinorotation. GUS staining was observed in cotyledons, leaves and hypocotyls of the seedlings (especially intense in vascular bundles), indicating intensive cellular processes with participation of this chaperone. Possible pathways of influence of clinorotation on HSP90-2 expression are discussed.

  14. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

    2016-01-01

    Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

  15. Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target.

    Directory of Open Access Journals (Sweden)

    Mariëlle I Gallegos Ruiz

    Full Text Available BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%, which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008, survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04. Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

  16. Evaluation of the toxic potential of calcium carbide in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9).

    Science.gov (United States)

    Danish, Mohd; Fatima, Ambreen; Khanam, Saba; Jyoti, Smita; Rahul; Ali, Fahad; Naz, Falaq; Siddique, Yasir Hasan

    2015-11-01

    In the present study the toxic potential of calcium carbide (CaC2) was studied on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). The third instar larvae were exposed to 2, 4, 8, 16 and 32×10(-3)g/ml of CaC2 in diet for 24h. The results reveal that the dose 2×10(-3)g/ml was not toxic but the remaining doses showed a dose dependent significant increase in the hsp70 expression, β-galactosidase activity, tissue damage, oxidative stress markers (lipid peroxidation and protein carbonyl content), glutathione-S-transferase activity, expression of Caspase 3 and 9, apoptotic index and DNA damage (midgut cells). A significant reduction as compared to control group in total protein, glutathione content and acetylcholinesterase activity was also observed. The Inductively Coupled Plasma Atomic Emission Spectroscopy analysis (ICPAES) reveals the presence of copper, iron, sodium, aluminium, manganese, calcium, nickel and mercury. The toxic effects of CaC2 in the present study may be attributed to the impurities present in it. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Model of Oxygen and Glucose Deprivation in PC12 Cells and Detection of HSP70 Protein

    Science.gov (United States)

    He, Jinting; Yang, Le; Shao, Yankun

    2018-01-01

    Objective: PC12 cell was used to set up a ischemia model by OGD and detected HSP70 protein. Methods: Use of PC12 cells induced by NGF stimulation into nerve cells, oxygen and glucose deprivation to build the nerve cells of oxygen and glucose deprivation model; using Western blot analysis of PC12 cells into neuron-like cells and oxygen-glucose deprivation model established. Results: The application of a final concentration of 50 ng / ml of NGF in DMEM complete mediumPC12 cells showed a typical neuronal morphology with the increase in cell culture time. NGF culture time showed a positive correlation, the establishment of oxygen and glucose deprivation (OGD) training environment, the OGD after nerve element appears different degrees of damage, OGD can effectively induce the expression of HSP70. Conclusion: PC12 cell transformed into cells by NGF; the cell model of OGD was established.

  18. Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

    Science.gov (United States)

    Antonietti, Patrick; Linder, Benedikt; Hehlgans, Stephanie; Mildenberger, Iris C; Burger, Michael C; Fulda, Simone; Steinbach, Joachim P; Gessler, Florian; Rödel, Franz; Mittelbronn, Michel; Kögel, Donat

    2017-01-01

    Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Effect of 3G cell phone exposure with computer controlled 2-D stepper motor on non-thermal activation of the hsp27/p38MAPK stress pathway in rat brain.

    Science.gov (United States)

    Kesari, Kavindra Kumar; Meena, Ramovatar; Nirala, Jayprakash; Kumar, Jitender; Verma, H N

    2014-03-01

    Cell phone radiation exposure and its biological interaction is the present concern of debate. Present study aimed to investigate the effect of 3G cell phone exposure with computer controlled 2-D stepper motor on 45-day-old male Wistar rat brain. Animals were exposed for 2 h a day for 60 days by using mobile phone with angular movement up to zero to 30°. The variation of the motor is restricted to 90° with respect to the horizontal plane, moving at a pre-determined rate of 2° per minute. Immediately after 60 days of exposure, animals were scarified and numbers of parameters (DNA double-strand break, micronuclei, caspase 3, apoptosis, DNA fragmentation, expression of stress-responsive genes) were performed. Result shows that microwave radiation emitted from 3G mobile phone significantly induced DNA strand breaks in brain. Meanwhile a significant increase in micronuclei, caspase 3 and apoptosis were also observed in exposed group (P 3G mobile phone exposure causes a transient increase in phosphorylation of hsp27, hsp70, and p38 mitogen-activated protein kinase (p38MAPK), which leads to mitochondrial dysfunction-mediated cytochrome c release and subsequent activation of caspases, involved in the process of radiation-induced apoptotic cell death. Study shows that the oxidative stress is the main factor which activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK is the pathway of principle stress response. Results conclude that 3G mobile phone radiations affect the brain function and cause several neurological disorders.

  20. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

    Science.gov (United States)

    Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

    2010-07-30

    Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

  1. The effect of glutamine infusion on the inflammatory response and HSP70 during human experimental endotoxaemia

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Pedersen-Skovsgaard, Theis; Mortensen, Ole Hartvig

    2009-01-01

    was associated with alterations in white blood cell and differential counts, tumour necrosis factor-alpha, IL-6, temperature and heart rate, but glutamine affected neither the endotoxin-induced change in these variables nor the expression of HSP70 in BMNCs. CONCLUSIONS: Endotoxin reduced plasma glutamine...

  2. Protein synthesis in TE 671/RD (human rabdomiosarcoma) cells treated with thapsigargin and hyperthermia: impairment of HSP 70 induction.

    Science.gov (United States)

    Delpino, A; Piselli, P; Mangano, G

    1995-01-01

    In this study we considered the quantitative and qualitative changes of protein synthetic activity occurring in TE 671/RD cells treated with thapsigargin (TG), with hyperthermia (HT) or with a combination of both these agents. In cells treated with TG (100 nM, continuous exposure), the overall protein synthetic activity was initially inhibited but subsequently recovered to about 60% of the initial level. Chronic TG exposure was also able to induce the expression of GRP 78. The rate of synthesis of GRP 78, after a lag period of about 2 h, increased gradually to reach a maximum (9-fold induction) after 6 h of TG-treatment and was then maintained at that level up to 18 h. A weak induction of GRP 94 was observed following 6-8 h of continuous exposure to TG. In cells treated with HT (43 degrees C for 30 min), a typical heat shock response was observed: in particular, the relative rate of synthesis of HSP 70 (the major heat-inducible mammalian heat shock protein) was increased 10-fold over the constitutive level. The heat-promoted induction of HSP 70 was significantly reduced by concomitant or previous exposure to TG. When TG and HT were administred simultaneously, the increase in HSP 70 synthesis was only 4.7-fold over the control level, while in cells pre-treated for 1 h with TG before the hyperthermic challenge the rate of HSP 70 synthesis was only stimulated 2-fold. In both these conditions, by contrast, it was apparent that HT did not affect the TG-promoted induction of GRP 78. The correlations between the TG-induced mobilization of cytosolic Ca2+ and the effects on protein synthesis are discussed.

  3. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    International Nuclear Information System (INIS)

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

    2006-01-01

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  4. Serum Heat Shock Protein 70 Concentration in Relation to Polycystic Ovary Syndrome in a Non-Obese Chinese Population.

    Directory of Open Access Journals (Sweden)

    Hui Gao

    Full Text Available Polycystic ovary syndrome (PCOS represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70 concentrations and PCOS in a non-obese Chinese population.Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI levels, as well as Insulin resistance index (HOMA-IR (P < 0.05. Identically, markers of oxidative stress (malondialdehyde (MDA, 8-Hydroxy-desoxyguanosine (8-OHdG, Nitric oxide (NO and inflammation (tumor necrosis factor-alpha (TNF-α, C-reactive protein (CRP were markedly increased when compared to controls (P < 0.05. Elevated serum Hsp70 was positively correlated with IR, oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI. The receiver-operating characteristic curve (ROC analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946 vs. 0.822 (95% CI 0.744-0.900; P for difference = 0.015.Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.

  5. Polymorphisms of heat shock protein 70 genes (HSPA1A, HSPA1B and HSPA1L and susceptibility of noise-induced hearing loss in a Chinese population: A case-control study.

    Directory of Open Access Journals (Sweden)

    Yanhong Li

    Full Text Available Noise-induced hearing loss (NIHL is the second-most frequent form of sensorineural hearing loss. When exposed to the same noise, some workers develop NIHL while others do not, suggesting that NIHL may be associated with genetic factors. To explore the relationship between single nucleotide polymorphisms (SNPs in heat shock protein 70 (HSP70 genes (HSPA1A, HSPA1B and HSPA1L and susceptibility to NIHL in Han Chinese workers exposed to noise, a case-control association study was carried out with 286 hearing loss cases and 286 matched with gender, age, type of work, and exposure time, drawn from a population of 3790 noise-exposed workers. Four SNPs were selected and genotyped. Subsequently, the effects of the alleles and genotypes of the three HSP70 genes (HSPA1A, HSPA1B and HSPA1L on NIHL were analyzed by using a conditional logistic regression. A generalized multiple dimensionality reduction (GMDR was applied to further detect an interaction between the four SNPs. Compared with the combined genotypes CC/TC, carriers of the TT genotype of rs2763979 appeared to show greater susceptibility to NIHL (P = 0.042, adjusted OR = 1.731, 95% CI 1.021-2.935. A significant interaction between rs2763979 and CNE was found (P = 0.029, and a significant association was found between TT of s2763979 and NIHL (P = 0.024, adjusted OR = 5.694, 95%CI 1.256-25.817 in the 96 dB (A≤CNE<101 dB (A group. The results suggest that the rs2763979 locus of the HSP70 genes may be associated with susceptibility to NIHL in Chinese individuals, and other HSP70 genes may also be susceptibility genes for NIHL, but the results must be further replicated in additional independent sample sets.

  6. Evidence-based efficacy of adaptogens in fatigue, and molecular mechanisms related to their stress-protective activity.

    Science.gov (United States)

    Panossian, Alexander; Wikman, Georg

    2009-09-01

    The aim of this review article is to assess the level of scientific evidence presented by clinical trials of adaptogens in fatigue, and to provide a rationale at the molecular level for verified effects. Strong scientific evidence is available for Rhodiola rosea SHR-5 extract, which improved attention, cognitive function and mental performance in fatigue and in chronic fatigue syndrome. Good scientific evidence has been documented in trails in which Schisandra chinensis and Eleutherococcus senticosus increased endurance and mental performance in patients with mild fatigue and weakness. Based on their efficacy in clinical studies, adaptogens can be defined as a pharmacological group of herbal preparations that increase tolerance to mental exhaustion and enhance attention and mental endurance in situations of decreased performance. The beneficial stress-protective effect of adaptogens is related to regulation of homeostasis via several mechanisms of action associated with the hypothalamic-pituitary-adrenal axis and the control of key mediators of stress response such as molecular chaperons (e.g. Hsp70), stress-activated c-Jun N-terminal protein kinase (JNK1), Forkhead Box O transcription factor DAF-16, cortisol and nitric oxide (NO). The key point of action of phytoadaptogens appears to be their up-regulating and stress-mimetic effects on the "stress-sensor" protein Hsp70, which plays an important role in cell survival and apoptosis. Hsp70 inhibits the expression of NO synthase II gene and interacts with glucocorticoid receptors directly and via the JNK pathway, thus affecting the levels of circulating cortisol and NO. Prevention of stress-induced increase in NO, and the associated decrease in ATP production, results in increased performance and endurance. Adaptogen-induced up-regulation of Hsp70 triggers stress-induced JNK-1 and DAF-16-mediated pathways regulating the resistance to stress and resulting in enhanced mental and physical performance and, possibly

  7. Silencing of Hsp27 and Hsp72 in glioma cells as a tool for programmed cell death induction upon temozolomide and quercetin treatment

    Energy Technology Data Exchange (ETDEWEB)

    Jakubowicz-Gil, Joanna, E-mail: jjgil@poczta.umcs.lublin.pl [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Langner, Ewa [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Bądziul, Dorota [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Wertel, Iwona [1st Department of Gynaecology, University School of Medicine, Staszica 16, 20-081 Lublin (Poland); Rzeski, Wojciech [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Department of Immunology and Virology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland)

    2013-12-15

    The aim of the present study was to investigate whether silencing of Hsp27 or Hsp72 expression in glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cells increases their sensitivity to programmed cell death induction upon temozolomide and/or quercetin treatment. Transfection with specific siRNA was performed for the Hsp gene silencing. As revealed by microscopic observation and flow cytometry, the inhibition of Hsp expression was correlated with severe apoptosis induction upon the drug treatment studied. No signs of autophagy were detected. This was correlated with a decreased mitochondrial membrane potential, increased level of cytochrome c in the cytoplasm, and activation of caspase 3 and caspase 9. All these results suggest that the apoptotic signal was mediated by an internal pathway. Additionally, in a large percentage of cells treated with temozolomide, with or without quercetin, granules within the ER system were found, which was accompanied by an increased level of caspase 12 expression. This might be correlated with ER stress. Quercetin and temozolomide also changed the shape of nuclei from circular to “croissant like” in both transfected cell lines. Our results indicate that blocking of Hsp27 and Hsp72 expression makes T98G cells and MOGGCCM cells extremely vulnerable to apoptosis induction upon temozolomide and quercetin treatment and that programmed cell death is initiated by an internal signal. - Highlights: • Hsps gene silencing induced severe apoptosis upon temozolomide–quercetin treatment • Apoptosis in transfected glioma cells was initiated by internal signal • Programmed cell death was preceded by ER stress • Temozolomide–quercetin treatment changed nuclei shape in transfected glioma cells.

  8. Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

    Science.gov (United States)

    Montalvo, Ana M; Fraga, Jorge; Tirado, Dídier; Blandón, Gustavo; Alba, Annia; Van der Auwera, Gert; Vélez, Iván Darío; Muskus, Carlos

    2017-07-01

    Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

  9. The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae).

    Science.gov (United States)

    Herrero, Óscar; Aquilino, Mónica; Sánchez-Argüello, Paloma; Planelló, Rosario

    2018-01-01

    Bisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled "BPA-free". Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 μg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.

  10. Circulating antibodies to inducible heat shock protein 70 in patients with uveitis

    NARCIS (Netherlands)

    de Smet, M. D.; Ramadan, A.

    2001-01-01

    Heat shock proteins with molecular weight 70 kDa (hsp70) are highly conserved immunogenic intracellular molecules. There are two main subtypes: one is expressed constitutively (hsc70), while the other is induced under stressful conditions (ihsp70). Using an ELISA directed against recombinant human

  11. Intracellular proteins produced by mammalian cells in response to environmental stress

    Science.gov (United States)

    Goochee, Charles F.; Passini, Cheryl A.

    1988-01-01

    The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

  12. Heat Shock Protein 70 Enhances Mucosal Immunity against Human Norovirus When Coexpressed from a Vesicular Stomatitis Virus Vector

    Science.gov (United States)

    Ma, Yuanmei; Duan, Yue; Wei, Yongwei; Liang, Xueya; Niewiesk, Stefan; Oglesbee, Michael

    2014-01-01

    ABSTRACT Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 106 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 106 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is

  13. Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

    Directory of Open Access Journals (Sweden)

    Wang Lin

    2010-04-01

    Full Text Available Abstract Background The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin on human retinal pigment epithelial cells is investigated. Methods MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. Results 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. Conclusions 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.

  14. HEATING AND ULTRAVIOLET LIGHT ACTIVATE ANTI-STRESS GENE FUNCTIONS IN HUMANS

    Directory of Open Access Journals (Sweden)

    Victor Fadeevitch Semenkov

    2015-07-01

    Full Text Available All types of cell stress are accompanied by the activation of anti-stress genes that can suppress ROS synthesis. We hypothesized that different environmental factors would affect organisms through the activation of anti-stress genes by autologous serum (AS proteins, followed by the synthesis of molecules that increase cell resistance to oxidative stress. The goal of this work was to study the influence of AS on ROS production by peripheral blood neutrophils isolated from donors in different age groups. Neutrophils were isolated from 59 donors (38-94 years old. AS was heated at 100˚C for 30 sec. or irradiated by ultraviolet light (UV at 200-280 nm and 8 W for 10 min. Neutrophils were exposed to heat shock at 42˚C for 1 min. (short-term heating stress or 43˚C for 10 min., followed by the determination of the chemiluminescence reaction induced by zymosan. AS can increase or decrease ROS production by neutrophils depending on the structure of the proteins in the serum; these structures can be changed by heating or UV treatment and the temperature of their interaction (4˚C or 37˚C. We propose that the effect of environmental factors on AS proteins can cause an adverse increase in oxidative stress levels due to the functional reduction of anti-stress genes. We found a negative correlation between the quantity of intracellular Hsp70 and levels of intracellular ROS production following 10 minutes of heat shock at 43°C. Short-term heating stress (1 minute at 42°C was followed by a prominent reduction in ROS production. This effect may be a result of the impact of the hormone adrenaline on the functions of anti-stress genes. Indeed, the same effect was observed after treatment of the neutrophils with adrenaline at concentrations of 10-4 M and 10-5 M. In contrast, dexamethasone from the other stress hormone group did not evoke the same effect at the same concentrations.

  15. Expression of three sHSP genes involved in heat pretreatment-induced chilling tolerance in banana fruit.

    Science.gov (United States)

    He, Li-hong; Chen, Jian-ye; Kuang, Jian-fei; Lu, Wang-jin

    2012-07-01

    Banana fruit is highly susceptible to chilling injury. In previous research it was shown that heat pretreatment of banana fruit at 38 °C for 3 days before storage at a chilling temperature of 8 °C for 12 days prevented increases in visible chilling injury index, electrolyte leakage and malondialdehyde content and also decreases in lightness and chroma, indicating that heat pretreatment could effectively alleviate chilling injury of banana fruit. However, little is known about the role of small heat shock proteins (sHSPs) in postharvest chilling tolerance of banana fruit. In the present study, three cytosolic sHSP expression profiles in peel and pulp tissues of banana fruit during heat pretreatment and subsequent chilled storage (8 °C) were investigated in relation to heat pretreatment-induced chilling tolerance. Three full-length cDNAs of cytosolic sHSP genes, including two class I sHSP (CI sHSP) and one class II sHSP (CII sHSP) cDNAs, named Ma-CI sHSP1, Ma-CI sHSP2 and Ma-CII sHSP3 respectively, were isolated and characterised from harvested banana fruit. Accumulation of Ma-CI sHSP1 mRNA transcripts in peel and pulp tissues and Ma-CII sHSP3 mRNA transcripts in peel tissue increased during heat pretreatment. Expression of all three Ma-sHSP genes in peel and pulp tissues was induced during subsequent chilled storage. Furthermore, Ma-CI sHSP1 and Ma-CII sHSP3 mRNA transcripts in pulp tissue and Ma-CI sHSP2 mRNA transcripts in peel and pulp tissues were obviously enhanced by heat pretreatment at days 6 and 9 of subsequent chilled storage. These results suggested that heat pretreatment enhanced the expression of Ma-sHSPs, which might be involved in heat pretreatment-induced chilling tolerance of banana fruit. Copyright © 2012 Society of Chemical Industry.

  16. A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in O. tsutsugamushi-Infected Human Macrophages.

    Directory of Open Access Journals (Sweden)

    Ching-Ying Wu

    Full Text Available Orientia (O. tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE inhibitors on TNF-α production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB, but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70 expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response.

  17. Cocaine withdrawal causes delayed dysregulation of stress genes in the hippocampus.

    Directory of Open Access Journals (Sweden)

    M Julia García-Fuster

    Full Text Available Relapse, even following an extended period of withdrawal, is a major challenge in substance abuse management. Delayed neurobiological effects of the drug during prolonged withdrawal likely contribute to sustained vulnerability to relapse. Stress is a major trigger of relapse, and the hippocampus regulates the magnitude and duration of stress responses. Recent work has implicated hippocampal plasticity in various aspects of substance abuse. We asked whether changes in stress regulatory mechanisms in the hippocampus may participate in the neuroadaptations that occur during prolonged withdrawal. We therefore examined changes in the rat stress system during the course of withdrawal from extended daily access (5-hours of cocaine self-administration, an animal model of addiction. Tissue was collected at 1, 14 and 28 days of withdrawal. Plasma corticosterone levels were determined and corticosteroid receptors (GR, MR, MR/GR mRNA ratios and expression of other stress-related molecules (HSP90AA1 and HSP90AB1 mRNA were measured in hippocampal subfields using in situ hybridization. Results showed a delayed emergence of dysregulation of stress genes in the posterior hippocampus following 28 days of cocaine withdrawal. This included increased GR mRNA in DG and CA3, increased MR and HSP90AA1 mRNA in DG, and decreased MR/GR mRNA ratio in DG and CA1. Corticosterone levels progressively decreased during the course of withdrawal, were normalized following 28 days of withdrawal, and were correlated negatively with GR and positively with MR/GR mRNA ratio in DG. These results suggest a role for the posterior hippocampus in the neuroadaptations that occur during prolonged withdrawal, and point to a signaling partner of GR, HSP90AA1, as a novel dysregulated target during cocaine withdrawal. These delayed neurobiological effects of extended cocaine exposure likely contribute to sustained vulnerability to relapse.

  18. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

    Directory of Open Access Journals (Sweden)

    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  19. Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

    Science.gov (United States)

    Golbabapour, Shahram; Gwaram, Nura Suleiman; Al-Obaidi, Mazen M Jamil; Soleimani, A F; Ali, Hapipah Mohd; Abdul Majid, Nazia

    2013-01-01

    Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP) 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg), the positive control (Tween 20 5% v/v, 5 mL/kg), and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg). After 1 h, all of the groups received ethanol 95% (5 mL/kg) but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg). The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E), immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  20. Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

    Science.gov (United States)

    Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

    2011-01-01

    Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

  1. Serum Heat Shock Protein 70 Concentration in Relation to Polycystic Ovary Syndrome in a Non-Obese Chinese Population.

    Science.gov (United States)

    Gao, Hui; Meng, Jie; Xu, Mengjing; Zhang, Shun; Ghose, Bishwajit; Liu, Jun; Yao, Ping; Yan, Hong; Wang, Di; Liu, Liegang

    2013-01-01

    Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70) concentrations and PCOS in a non-obese Chinese population. Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI) levels, as well as Insulin resistance index (HOMA-IR) (P oxidative stress (malondialdehyde (MDA), 8-Hydroxy-desoxyguanosine (8-OHdG), Nitric oxide (NO)) and inflammation (tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)) were markedly increased when compared to controls (P oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI). The receiver-operating characteristic curve (ROC) analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946) vs. 0.822 (95% CI 0.744-0.900); P for difference = 0.015). Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.

  2. Alteration of cytochrome P450 1 regulation and HSP 70 level in brain of juvenile common carp (Cyprinus carpio) after chronic exposure to tributyltin.

    Science.gov (United States)

    Li, Zhi-Hua; Zhong, Li-Qiao; Wu, Yan-Hua; Mu, Wei-Na

    2016-02-01

    Tributyltin (TBT), a toxic contaminant in aquatic environments, has bio-accumulated in aquatic food webs throughout the world and can be found at toxic levels in some biota. However, the molecular mechanisms and effects of TBT are not fully understood. The aim of the present study was to investigate the effect of long-term exposure of TBT on cytochrome P450 (CYP450) 1 regulation and heat-shock proteins (HSPs) profiling in brain of freshwater teleost. The effects of long-term exposure to TBT on mRNA expression of cytochrome P450 (CYP450) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in the brain of common carp were evaluated, as well as HSP 70 level. Fish were exposed to sublethal concentrations of TBT (75 ng/L, 0.75 μg/L and 7.5 μg/L) for 15, 30, and 60 days. Based on the results, long-term exposure (more than 15 days) to TBT could lead to obvious physiological-biochemical responses (based on EROD activity, HSP 70 level and CYP450 1 family genes expression). The mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. Thus, the measured physiological responses in fish brain could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity and could be used as potential biomarkers for monitoring the TBT pollution in the field.

  3. Role of heat shock protein Hsp25 in the response of the orofacial nuclei motor system to physiological stress

    Science.gov (United States)

    Murashov, A. K.; Talebian, S.; Wolgemuth, D. J.

    1998-01-01

    Although expression of the small heat shock protein family member Hsp25 has been previously observed in the central nervous system (CNS), both constitutively and upon induction, its function in the CNS remains far from clear. In the present study we have characterized the spatial pattern of expression of Hsp25 in the normal adult mouse brain as well as the changes in expression patterns induced by subjecting mice to experimental hyperthermia or hypoxia. Immunohistochemical analysis revealed a surprisingly restricted pattern of constitutive expression of Hsp25 in the brain, limited to the facial, trigeminal, ambiguus, hypoglossal and vagal motor nuclei of the brainstem. After hyperthermia or hypoxia treatment, significant increases in the levels of Hsp25 were observed in these same areas and also in fibers of the facial and trigeminal nerve tracts. Immunoblot analysis of protein lysates from brainstem also showed the same pattern of induction of Hsp25. Surprisingly, no other area in the brain showed expression of Hsp25, in either control or stressed animals. The highly restricted expression of Hsp25 implies that this protein may have a specific physiological role in the orofacial motor nuclei, which govern precise coordination between muscles of mastication and the pharynx, larynx, and face. Its rapid induction after stress further suggests that Hsp25 may serve as a specific molecular chaperone in the lower cholinergic motor neurons and along their fibers under conditions of stress or injury. Copyright 1998 Elsevier Science B.V.

  4. Complexity of rice Hsp100 gene family: lessons from rice genome ...

    Indian Academy of Sciences (India)

    Madhu Sudhan

    2007-03-29

    Mar 29, 2007 ... Chaperonins are a class of molecular chaperones found in prokaryotes and in the ... Keywords. Chaperone, gene family, Hsp100, Oryza sativa ..... Sculpting the proteome with AAA+ proteases and disassembly machines; Cell ...

  5. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-07-08

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Mirzaei

    2016-02-01

    Full Text Available Objective(s: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. Materials and Methods: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma, 5637 (bladder tumor and U-87MG (brain tumor cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our results indicated that fifteen genes (from 36 studied genes were down-regulated and two genes (DNAJC11 and DNAJC5B were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes showed different expressional pattern (up or down-expression based on tumor cell lines. Conclusion: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

  7. Analysis of the expression of putative heat-stress related genes in relation to thermotolerance of cork oak.

    Science.gov (United States)

    Correia, Barbara; Rodriguez, José Luis; Valledor, Luis; Almeida, Tânia; Santos, Conceição; Cañal, Maria Jesús; Pinto, Glória

    2014-03-15

    Cork oak (Quercus suber L.) is a research priority in the Mediterranean area and because of cork oaks' distribution these stands are experiencing daily stress. Based on projections of intensifying climate change and considering the key role of exploring the recovery abilities, cork oak seedlings were subjected to a cumulative temperature increase from 25°C to 55°C and subsequent recovery. CO2 assimilation rate, chlorophyll fluorescence, anthocyanins, proline and lipid peroxidation were used to evaluate plant performance, while the relative abundance of seven genes encoding for proteins of cork oak with a putative role in thermal/stress regulation (POX1, POX2, HSP10.4, HSP17a.22, CHS, MTL and RBC) was analyzed by qPCR (quantitative Polymerase Chain Reaction). A temperature change to 35°C showed abundance alterations in the tested genes; at 45°C, the molecular changes were associated with an antioxidant response, possibly modulated by anthocyanins. At 55°C, HSP17a.22, MTL and proline accumulation were evident. After recovery, physiological balance was restored, whereas POX1, HSP10.4 and MTL abundances were suggested to be involved in increased thermotolerance. The data presented here are expected to pinpoint some pathways changes occurring during such stress and further recovery in this particular Mediterranean species. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Characterization of heat shock cognate protein 70 gene and its differential expression in response to thermal stress between two wing morphs of Nilaparvata lugens (Stål).

    Science.gov (United States)

    Lu, Kai; Chen, Xia; Liu, Wenting; Zhou, Qiang

    2016-09-01

    Previous studies have demonstrated differences in thermotolerance between two wing morphs of Nilaparvata lugens, the most serious pest of rice across the Asia. To reveal the molecular regulatory mechanisms underlying the differential thermal resistance abilities between two wing morphs, a full-length of transcript encoding heat shock cognate protein 70 (Hsc70) was cloned, and its expression patterns across temperature gradients were analyzed. The results showed that the expression levels of NlHsc70 in macropters increased dramatically after heat shock from 32 to 38°C, while NlHsc70 transcripts in brachypters remained constant under different temperature stress conditions. In addition, NlHsc70 expression in the macropters was significantly higher than that in brachypters at 1 and 2h recovery from 40°C heat shock. There was no significant difference in NlHsc70 mRNA expression between brachypters and macropters under cold shock conditions. Therefore, NlHsc70 was indeed a constitutively expressed member of the Hsp70 family in brachypters of N. lugens, while it was heat-inducible in macropters. Furthermore, the survival rates of both morphs injected with NlHsc70 dsRNA were significantly decreased following heat shock at 40°C or cold shock at 0°C for 1h. These results suggested that the up-regulation of NlHsc70 is possibly related to the thermal resistance, and the more effective inducement expression of NlHsc70 in macropters promotes a greater thermal tolerance under temperature stress conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses, Hepatic Gene Expression, and Stress Protein Profiling.

    Science.gov (United States)

    Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

    2016-02-01

    In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na(+) -K(+) -ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation-related genes expression and stress-related proteins profiling in fish liver. Oxidative stress indices and Na(+) -K(+) -ATPase showed tissues-specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF-I axis and ion-regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose-dependent manner under TBT stress. In short, TBT-induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long-term experiments at lower concentrations will be necessary in the future. © 2015 Wiley Periodicals, Inc.

  10. Characteristics of six small heat shock protein genes from Bactrocera dorsalis: Diverse expression under conditions of thermal stress and normal growth.

    Science.gov (United States)

    Dou, Wei; Tian, Yi; Liu, Hong; Shi, Yan; Smagghe, Guy; Wang, Jin-Jun

    2017-11-01

    To explore the functions of small heat shock proteins (sHsps) in relation to thermal stress and development in Bactrocera dorsalis (Hendel), one of the most economically important pest species attacking a wide range of fruits and vegetables, six full-length cDNAs of sHsp genes (BdHsp17.7, 18.4, 20.4, 20.6, 21.6 and 23.8) were cloned, and the expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) exposures, were examined using real time quantitative PCR. The open reading frames (ORFs) of six sHsps are 453, 489, 537, 543, 567 and 630bp in length, encoding proteins with molecular weights of 17.7, 18.4, 20.4, 20.6, 21.6 and 23.8kDa, respectively. BdHsp18.4 and BdHsp20.4 maintained lower expression levels in both eggs and larvae, whereas remarkably up-regulated after the larval-pupal transformation, suggesting that these two sHsps may be involved in metamorphosis. Significant tissue specificity exists among sHsps: the highest expression of BdHsp20.6 and BdHsp23.8 in the Malpighian tubules and ovary, respectively, versus a peak in the fat body for others. BdHsp20.4 and BdHsp20.6 were significantly up-regulated by thermal stress. In contrast, BdHsp18.4 and BdHsp23.8 reacted only to heat stress. BdHsp17.7 and BdHsp21.6 were insensitive to both heat and cold stresses. The degree of sHsps response depends on intensity of 20E treatment, i.e., dose and time. These results strongly suggest functional differentiation within the sHsp subfamily in B. dorsalis. The physiological function of sHsp members under thermal stress and normal growth remains the subjects of further investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Vitrification affects nuclear maturation and gene expression of immature human oocytes

    Directory of Open Access Journals (Sweden)

    Abbas Shahedi

    2017-02-01

    Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

  12. The Hsp90 co-chaperones Sti1, Aha1, and P23 regulate adaptive responses to antifungal azoles

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    Xiaokui Gu

    2016-10-01

    Full Text Available Heat Shock Protein 90 (Hsp90 is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast, Aha1, and P23 (Sba1 in yeast were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

  13. Stressing Out Hsp90 in Neurotoxic Proteinopathies.

    Science.gov (United States)

    Inda, Carmen; Bolaender, Alexander; Wang, Tai; Gandu, Srinivasa R; Koren, John

    2016-01-01

    A toxic accumulation of proteins is the hallmark pathology of several neurodegenerative disorders. Protein accumulation is regularly prevented by the network of molecular chaperone proteins, including and especially Hsp90. For reasons not yet elucidated, Hsp90 and the molecular chaperones interact with, but do not degrade, these toxic proteins resulting in the pathogenic accumulation of proteins such as tau, in Alzheimer's Disease, and α-synuclein, in Parkinson's Disease. In this review, we describe the associations between Hsp90 and the pathogenic and driver proteins of several neurodegenerative disorders. We additionally describe how the inhibition of Hsp90 promotes the degradation of both mutant and pathogenic protein species in models of neurodegenerative diseases. We also examine the current state of Hsp90 inhibitors capable of crossing the blood-brain barrier; compounds which may be capable of slowing, preventing, and possible reversing neurodegenerative diseases.

  14. Fluoro-edenite Fibers Induce Expression of Hsp70 and Inflammatory Response

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    Michael Balazy

    2007-09-01

    Full Text Available Many asbestos-like mineral fibers have been detected in the air of mountainous and volcanic areas of Italy and other parts of the world. These fibers have been suspected to be the cause of increased incidences of lung cancer and other lung diseases in these areas. However, the mechanisms of the cellular response and defense following exposure to these microscopic fibers have not been characterized. We continue to study these mechanisms to be able to propose preventive strategies in large populations. The objective of the present study was to determine comparatively biological responses of mesothelial Met-5A and monocyte-macrophage J774 cells following exposure to two types of fluoro-edenite fibers having low and high iron content (labeled 19 and 27, respectively obtained from Biancavilla (Sicily, Italy. The reference fiber was a non-iron fibrous tremolite from Val di Susa (Piemonte, Italy. The cells were treated with 5, 50, and 100 μg of fibrous matter per 1 ml for 72 hr. We identified several key mechanisms by which cells responded and counteracted the injury induced by these fibers. The fibers caused induction of the heat shock protein 70 (Hsp70, stimulated formation of reactive oxygen species (detected by using DCFH-DA as a fluorescent probe and NO• (measured as nitrite. Exposure of cells to the fibers induced lactate dehydrogenase activity and decreased viability. The fluoro-endenite type 27 was the most potent fiber tested, which indicated that iron and possibly manganese contribute significantly to this fiber toxicity. The J774 cells were more sensitive to fluoro-edenite than Met-5A cells suggesting that the primary site of the fiberinduced inflammatory response could be the macrophage rather than the pulmonary epithelium. Fluoro-edenite produces more biological alterations with respect to non-iron tremolite. Hsp70 and free radicals could be important factors in the context of mineral fiber-induced acute lung injury

  15. The 70 kDa heat shock protein assists during the repair of chilling injury in the insect, Pyrrhocoris apterus.

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    Vladimír Kostál

    Full Text Available BACKGROUND: The Pyrrhocoris apterus (Insecta: Heteroptera adults attain high levels of cold tolerance during their overwintering diapause. Non-diapause reproducing adults, however, lack the capacity to express a whole array of cold-tolerance adaptations and show relatively low survival when exposed to sub-zero temperatures. We assessed the competence of non-diapause males of P. apterus for responding to heat- and cold-stresses by up-regulation of 70 kDa heat shock proteins (Hsps and the role of Hsps during repair of heat- and cold-induced injury. PRINCIPAL FINDINGS: The fragments of P. apterus homologues of Hsp70 inducible (PaHsp70 and cognate forms (PaHsc70 were cloned and sequenced. The abundance of mRNA transcripts for the inducible form (qPCR and corresponding protein (Western blotting were significantly up-regulated in response to high and low temperature stimuli. In the cognate form, mRNA was slightly up-regulated in response to both stressors but very low or no up-regulation of protein was apparent after heat- or cold-stress, respectively. Injection of 695 bp-long Pahsp70 dsRNA (RNAi caused drastic suppression of the heat- and cold-stress-induced Pahsp70 mRNA response and the up-regulation of corresponding protein was practically eliminated. Our RNAi predictably prevented recovery from heat shock and, in addition, negatively influenced repair of chilling injuries caused by cold stress. Cold tolerance increased when the insects were first exposed to a mild heat shock, in order to trigger the up-regulation of PaHsp70, and subsequently exposed to cold stress. CONCLUSION: Our results suggest that accumulation of PaHsp70 belongs to a complex cold tolerance adaptation in the insect Pyrrhocoris apterus.

  16. Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential

    DEFF Research Database (Denmark)

    Bross, Peter; Li, Zhijie; Hansen, Jakob

    2007-01-01

    for variations in the HSPD1 and HSPE1 genes encoding the mitochondrial Hsp60/Hsp10 chaperone complex: two patients with multiple mitochondrial enzyme deficiency, 61 sudden infant death syndrome cases (MIM: #272120), and 60 patients presenting with ethylmalonic aciduria carrying non-synonymous susceptibility...... variations in the ACADS gene (MIM: *606885 and #201470). Besides previously reported variations we detected six novel variations: two in the bidirectional promoter region, and one synonymous and three non-synonymous variations in the HSPD1 coding region. One of the non-synonymous variations was polymorphic...... in patient and control samples, and the rare variations were each only found in single patients and absent in 100 control chromosomes. Functional investigation of the effects of the variations in the promoter region and the non-synonymous variations in the coding region indicated that none of them had...

  17. Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

    DEFF Research Database (Denmark)

    Nylandsted, J; Jäättelä, M; Hoffmann, E K

    2004-01-01

    Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

  18. Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

    Energy Technology Data Exchange (ETDEWEB)

    Worrall, Liam; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [School of Biological Sciences, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom)

    2006-09-01

    Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

  19. Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.

    Science.gov (United States)

    Deville, Célia; Carroni, Marta; Franke, Kamila B; Topf, Maya; Bukau, Bernd; Mogk, Axel; Saibil, Helen R

    2017-08-01

    Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo-electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5'- O -(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.

  20. Identification of multiple small heat-shock protein genes in Plutella xylostella (L.) and their expression profiles in response to abiotic stresses.

    Science.gov (United States)

    Chen, Xi'en; Zhang, Yalin

    2015-01-01

    We identify and characterize 14 small heat-shock protein (sHSP) genes from the diamondback moth (DBM), Plutella xylostella (L.), a destructive pest. Phylogenetic analyses indicate that, except for sHSP18.8 and sHSP19.22, the other 12 DBM sHSPs belong to five known insect sHSP groups. Developmental expression analysis revealed that most sHSPs peaked in the pupal and adult stages. The transcripts of sHSPs display tissue specificity with two exhibiting constitutive expression in four tested tissues. Expression of sHSP18.8 in fourth instar larvae is not induced by the tested abiotic stressors, and unless sHSP21.8 is not sensitive to thermal stress, 12 sHSPs are significantly up-regulated. The messenger RNA (mRNA) levels of all sHSPs are reduced under oxidative stress. Food deprivation leads to significant down-regulation of three sHSPs. The majority of sHSPs show expression variation to various heavy metals, whereas mRNA abundances of sHSP22.1 and sHSP 28.9 are reduced by four heavy metals. The responses of sHSPs to indoxacarb and cantharidin are varied. Beta-cypermethrin and chlorfenapyr exposure results in an increase of 13 sHSP transcripts and a reduction of 12 sHSP transcripts, respectively. These results show that different sHSPs might play distinct roles in the development and regulation of physiological activities, as well as in response to various abiotic stresses of DBM.

  1. Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

    Science.gov (United States)

    Zhadobov, M; Sauleau, R; Le Coq, L; Debure, L; Thouroude, D; Michel, D; Le Dréan, Y

    2007-04-01

    This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins. (c) 2006 Wiley-Liss, Inc.

  2. The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

    NARCIS (Netherlands)

    Wimmer, Bernhard; Lottspeich, Friedrich; Klei, Ida van der; Veenhuis, Marten; Gietl, Christine

    1997-01-01

    The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons with molecular masses of 70 and 72 KDa, Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of

  3. dFOXO Activates Large and Small Heat Shock Protein Genes in Response to Oxidative Stress to Maintain Proteostasis in Drosophila.

    Science.gov (United States)

    Donovan, Marissa R; Marr, Michael T

    2016-09-02

    Maintaining protein homeostasis is critical for survival at the cellular and organismal level (Morimoto, R. I. (2011) Cold Spring Harb. Symp. Quant. Biol. 76, 91-99). Cells express a family of molecular chaperones, the heat shock proteins, during times of oxidative stress to protect against proteotoxicity. We have identified a second stress responsive transcription factor, dFOXO, that works alongside the heat shock transcription factor to activate transcription of both the small heat shock protein and the large heat shock protein genes. This expression likely protects cells from protein misfolding associated with oxidative stress. Here we identify the regions of the Hsp70 promoter essential for FOXO-dependent transcription using in vitro methods and find a physiological role for FOXO-dependent expression of heat shock proteins in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Genetic diversity of the 3ꞌ and 5ꞌ untranslated regions of the ...

    African Journals Online (AJOL)

    Heat stress proteins are important factors in protecting cells against environmental stress. The HSP70.1 gene is one of the most important members of the heat stress protein family, which is essential for life, production and reproduction. In this study, partial regions of HSP70.1 (3ꞌ and 5ꞌ untranslated regions (UTRs)) were ...

  5. Sequence characterization of heat shock protein gene of Cyclospora cayetanensis isolates from Nepal, Mexico, and Peru.

    Science.gov (United States)

    Sulaiman, Irshad M; Torres, Patricia; Simpson, Steven; Kerdahi, Khalil; Ortega, Ynes

    2013-04-01

    We have described the development of a 2-step nested PCR protocol based on the characterization of the 70-kDa heat shock protein (HSP70) gene for rapid detection of the human-pathogenic Cyclospora cayetanensis parasite. We tested and validated these newly designed primer sets by PCR amplification followed by nucleotide sequencing of PCR-amplified HSP70 fragments belonging to 16 human C. cayetanensis isolates from 3 different endemic regions that include Nepal, Mexico, and Peru. No genetic polymorphism was observed among the isolates at the characterized regions of the HSP70 locus. This newly developed HSP70 gene-based nested PCR protocol provides another useful genetic marker for the rapid detection of C. cayetanensis in the future.

  6. Expression of HSPs: an adaptive mechanism during long-term heat stress in goats ( Capra hircus)

    Science.gov (United States)

    Dangi, Satyaveer Singh; Gupta, Mahesh; Dangi, Saroj K.; Chouhan, Vikrant Singh; Maurya, V. P.; Kumar, Puneet; Singh, Gyanendra; Sarkar, Mihir

    2015-08-01

    Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups ( n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest ( P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest ( P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

  7. UV-B radiation-induced oxidative stress and p38 signaling pathway involvement in the benthic copepod Tigriopus japonicus.

    Science.gov (United States)

    Kim, Bo-Mi; Rhee, Jae-Sung; Lee, Kyun-Woo; Kim, Min-Jung; Shin, Kyung-Hoon; Lee, Su-Jae; Lee, Young-Mi; Lee, Jae-Seong

    2015-01-01

    Ultraviolet B (UV-B) radiation presents an environmental hazard to aquatic organisms. To understand the molecular responses of the intertidal copepod Tigriopus japonicus to UV-B radiation, we measured the acute toxicity response to 96 h of UV-B radiation, and we also assessed the intracellular reactive oxygen species (ROS) levels, glutathione (GSH) content, and antioxidant enzyme (GST, GR, GPx, and SOD) activities after 24 h of exposure to UV-B with LD50 and half LD50 values. Also, expression patterns of p53 and hsp gene families with phosphorylation of p38 MAPK were investigated in UV-B-exposed copepods. We found that the ROS level, GSH content, and antioxidant enzyme activity levels were increased with the transcriptional upregulation of antioxidant-related genes, indicating that UV-B induces oxidative stress by generating ROS and stimulating antioxidant enzymatic activity as a defense mechanism. Additionally, we found that p53 expression was significantly increased after UV-B irradiation due to increases in the phosphorylation of the stress-responsive p38 MAPK, indicating that UV-B may be responsible for inducing DNA damage in T. japonicus. Of the hsp family genes, transcriptional levels of hsp20, hsp20.7, hsp70, and hsp90 were elevated in response to a low dose of UV-B radiation (9 kJ m(-2)), suggesting that these hsp genes may be involved in cellular protection against UV-B radiation. In this paper, we performed a pathway-oriented mechanistic analysis in response to UV-B radiation, and this analysis provides a better understanding of the effects of UV-B in the intertidal benthic copepod T. japonicus. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Interpopulation differences in expression of candidate genes for salinity tolerance in winter migrating anadromous brown trout (Salmo trutta L.

    Directory of Open Access Journals (Sweden)

    Thomsen Dennis S

    2008-01-01

    Full Text Available Abstract Background Winter migration of immature brown trout (Salmo trutta into freshwater rivers has been hypothesized to result from physiologically stressful combinations of high salinity and low temperature in the sea. Results We sampled brown trout from two Danish populations entering different saline conditions and quantified expression of the hsp70 and Na/K-ATPases α 1b genes following acclimation to freshwater and full-strength seawater at 2°C and 10°C. An interaction effect of low temperature and high salinity on expression of both hsp70 and Na/K-ATPase α 1b was found in trout from the river entering high saline conditions, while a temperature independent up-regulation of both genes in full-strength seawater was found for trout entering marine conditions with lower salinities. Conclusion Overall our results support the hypothesis that physiologically stressful conditions in the sea drive sea-run brown trout into freshwater rivers in winter. However, our results also demonstrate intra-specific differences in expression of important stress and osmoregulative genes most likely reflecting adaptive differences between trout populations on a regional scale, thus strongly suggesting local adaptations driven by the local marine environment.

  9. Impact of short-term heat stress on physiological responses and expression profile of HSPs in Barbari goats

    Science.gov (United States)

    Dangi, Satyaveer Singh; Gupta, Mahesh; Nagar, Vimla; Yadav, Vijay Pratap; Dangi, Saroj K.; Shankar, Om; Chouhan, Vikrant Singh; Kumar, Puneet; Singh, Gyanendra; Sarkar, Mihir

    2014-12-01

    Six, nonpregnant, Barbari goats aged 4-5 years were selected for the study. For the first 6 days, the animals were kept in psychrometric chamber at thermoneutral temperature for 6 h each day to make them acclimated to climatic chamber. On the 7th day, the animals were exposed to 41 °C temperature for 3 h and then to 45 °C for the next 3 h. Cardinal physiological responses were measured, and blood samples (3 ml) were collected at 1-h interval during the heat exposure period and then once after 6 h of the heat exposure. The rectal temperature (RT) and respiratory rate (RR) increased significantly ( P < 0.05) during the heat exposure compared to pre- and postexposure. The relative messenger RNA (mRNA) expression of heat shock protein (HSP)60, HSP70, and HSP90 increased significantly ( P < 0.05) within 1 h after exposure to heat stress at 41 and 45 °C and decreased significantly ( P < 0.05) in next 2 h but remain significantly ( P < 0.05) elevated from preexposure. HSP105/110 relative mRNA expression level remained unchanged during the first 4 h, and thereafter, it increased significantly ( P < 0.05) and reached the peak at 6 h. Relative protein expression pattern of HSPs during exposure to heat stress showed similar trend as observed for the relative mRNA expression. Given the response sensitivity and intensity of HSP genes to environmental stresses, HSP70 was found to be the most sensitive to temperature fluctuation, and it could be used as an important molecular biomarker to heat stress in animals.

  10. The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae.

    Directory of Open Access Journals (Sweden)

    Óscar Herrero

    Full Text Available Bisphenol S (BPS is an industrial alternative to the endocrine disruptor bisphenol A (BPA, and can be found in many products labeled "BPA-free". Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1 crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3 that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13 which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 μg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control were EcR (3.8, ERR (2, E74 (2.4, cyp18a1 (2.5, hsp70 (1.7, hsp40 (2.5, cyp4g (6.4, GPx (1.8, and GST (2.1, while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.

  11. Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome.

    Directory of Open Access Journals (Sweden)

    Melody S Clark

    Full Text Available BACKGROUND: The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. METHODOLOGY/PRINCIPAL FINDINGS: The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp. In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS, providing a resource for population and also gene function studies. CONCLUSIONS: This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding

  12. Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

    Science.gov (United States)

    Salces-Ortiz, Judit; Ramón, Manuel; González, Carmen; Pérez-Guzmán, M Dolores; Garde, J Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H; Serrano, M Magdalena

    2015-01-01

    Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and

  13. Enantioselective effect of bifenthrin on antioxidant enzyme gene expression and stress protein response in PC12 cells.

    Science.gov (United States)

    Lu, Xianting

    2013-07-01

    Enantioselectivity in toxicology and the health risk of chiral xenobiotics have become frontier topics interfacing chemistry and toxicology. Our previous results showed that cis-bifenthrin (cis-BF) induced cytotoxicity and apoptosis in vitro in an enantioselective manner. However, the exact molecular mechanisms of synthetic pyrethroid-induced enantioselective apoptosis and cytotoxicity have so far received limited research attention. In the present study, the expression patterns of different genes encoding heat shock protein and antioxidant enzymes were investigated by real-time quantitative PCR in rat adrenal pheochromocytoma (PC12) cells after exposure to cis-BF and its enantiomers. The results showed that exposure to 1S-cis-BF resulted in increased transcription of HSP90, HSP70, HSP60, Cu-Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione-s-transferase at a concentration of 5 µm and above, while exposure to 1R-cis-BF and rac-cis-BF exhibited these effects to lesser degrees. In addition, induction of antioxidant enzyme gene expression produced by 1S-cis-BF might occur, at least in part, through activation of p38 mitogen-activated protein kinases (MAPK) and extracellular regulated kinases, while increase in stress protein response produced by 1S-cis-BF might occur through the p38 MAPK signaling pathway. The results not only suggest that enantioselectivity should be considered in evaluating the ecotoxicological effects and health risk of chiral contaminants, but also will improve the understanding of molecular mechanism for chiral chemical-induced cytotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.

  14. Influence of the cestode Ligula intestinalis and the acanthocephalan Polymorphus minutus on levels of heat shock proteins (HSP70) and metallothioneins in their fish and crustacean intermediate hosts

    International Nuclear Information System (INIS)

    Frank, Sabrina N.; Godehardt, Saskia; Nachev, Milen; Trubiroha, Achim; Kloas, Werner; Sures, Bernd

    2013-01-01

    It is a common method to analyse physiological mechanisms of organisms – commonly referred to as biomarkers – to indicate the presence of environmental pollutants. However, as biomarkers respond to a wide range of stressors we want to direct the attention on natural stressors, i.e. on parasites. After two years maintenance under controlled conditions, roach (Rutilus rutilus) revealed no influence on levels of metallothionein by the parasite Ligula intestinalis. The same was found for Gammarus fossarum infected with Polymorphus minutus. However, the heat shock protein (HSP70) response was affected in both host-parasite systems. While the infection of roach resulted in reduced levels of HSP70 compared to uninfected roach, the infection in G. fossarum led to higher levels of HSP70. We also analysed the effect of a 14 days Cd exposure (4 μg/L) on the uninfected and infected gammarids. The exposure resulted in induced levels for both, metallothionein and HSP70 whereas the combination of stressors, parasite and exposure, revealed a decrease for levels of HSP70 in comparison to the metal exposure only. Accordingly, parasites as natural parts of aquatic ecosystems have to be considered in ecotoxicological research. -- Highlights: •We show how parasites and pollutant affect biomarkers. •Metallothioneins were not influenced by parasites. •Heat shock proteins are modulated by parasites. •Biomarker levels of organisms are dependent on infection status. •Infection with parasites has to be considered in ecotoxicology. -- Parasites are capable of affecting host physiology and therefore modulate biomarker responses

  15. Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

    Directory of Open Access Journals (Sweden)

    Gabrielle Stetz

    2017-01-01

    Full Text Available Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of

  16. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows.

    Science.gov (United States)

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-06-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (Pdairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat stress in dairy cows in the future.

  17. Pre-treatment with mild whole-body heating prevents gastric ulcer induced by restraint and water-immersion stress in rats.

    Science.gov (United States)

    Itoh, Y H; Noguchi, R

    2000-01-01

    The purpose of this study was to assess the preventive effect of pre-mild whole-body heating (WBH) on gastric ulcer induced by restraint and water-immersion stress. The ulcer index and ulcer area ratio in rats exposed to restraint and water-immersion stress were significantly decreased (p immersion stress alone (p immersion, thereby preventing gastric ulcer formation. Pre-treatment with mild WBH is the safest cytoprotective method through the accumulation of HSP 70f. The concentration of HSP 70f in peripheral lymphocytes may be a useful clinical laboratory indicator for assessing the level of HSP 70f as having cytoprotective activity.

  18. Effect of moderate hypoxia at three acclimation temperatures on stress responses in Atlantic cod with different haemoglobin types

    DEFF Research Database (Denmark)

    Methling, Caroline; Aluru, Neelakanteswar; Vijayan, Mathilakath M

    2010-01-01

    in a difference in stress response to hypoxia exposure. Two hsp70-isoforms (labelled a and b) were detected and they differed in expression in the gills but not in the liver of Atlantic cod. Acclimation temperature significantly affected the expression of hsp70 in the liver, and in an isoform-specific manner...... in the gills. Hypoxia exposure increased the expression of hsp70 in the liver, but not the gills, of cod and this response was not influenced by the acclimation temperature. The expression of hsp70 in both tissues did not differ between fish with different haemoglobin types. Acclimation temperature...... hypoxic exposure influence the organismal and cellular stress responses in Atlantic cod. We hypothesise that HbI-2 fish are more tolerant to short-term hypoxic episodes than HbI-1 fish, and this adaptation may be independent of tissue hsp70 expression....

  19. Oxidative stress and gene expression of earthworm (Eisenia fetida) to clothianidin.

    Science.gov (United States)

    Liu, Tong; Wang, Xiuguo; You, Xiangwei; Chen, Dan; Li, Yiqiang; Wang, Fenglong

    2017-08-01

    Neonicotinoid insecticides have become the most widely used pesticides in the world. Clothianidin is a novel neonicotinoid insecticide with a thiazolyl ring that exhibits excellent biological efficacy against a variety of pests. In the present study, the oxidative stress and genotoxicity of clothianidin on earthworms were evaluated. Moreover, the effective concentrations of clothianidin in artificial soil were monitored during the whole exposure period. The results showed that clothianidin was stable in artificial soil and that the residue concentrations were 0.094, 0.476, and 0.941mg/kg after 28 d of exposure, which represented changes no more than 10% compared to the concentrations on the 0th day. Additionally, both the concentration of and exposure time to clothianidin had a substantial influence on biomarkers in earthworms. At 0.5mg/kg and 1.0mg/kg, the reactive oxygen species (ROS) levels were greatly enhanced, causing changes in antioxidant enzyme activities, damage to biological macromolecules and abnormal expression of functional genes. Additionally, the present results showed that superoxide dismutase (SOD), DNA damage and heat shock protein 70 (HSP70) may be good indicators for environmental risk assessment of clothianidin to earthworms. Copyright © 2017. Published by Elsevier Inc.

  20. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

    Energy Technology Data Exchange (ETDEWEB)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito, E-mail: y-ihara@wakayama-med.ac.jp

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

  1. HSP90 Shapes the Consequences of Human Genetic Variation.

    Science.gov (United States)

    Karras, Georgios I; Yi, Song; Sahni, Nidhi; Fischer, Máté; Xie, Jenny; Vidal, Marc; D'Andrea, Alan D; Whitesell, Luke; Lindquist, Susan

    2017-02-23

    HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Circulating Heat Shock Protein 70 in Health, Aging and Disease

    Directory of Open Access Journals (Sweden)

    Demanet Christian

    2011-03-01

    Full Text Available Abstract Background Heat shock proteins (Hsp are ubiquitously synthesised in virtually all species and it is hypothesised that they might have beneficial health effects. Recent studies have identified circulating Hsp as an important mediator in inflammation - the effects of low-grade inflammation in the aging process are overwhelming. While much is known about intracellular Hsp70, scant data exist on circulating Hsp70 in the aging context. Therefore, the objectives of this study were to investigate the effect of age and disease on circulating Hsp70 and, in particular, to evaluate the association between circulating Hsp70 and inflammatory parameters. Results Serum Hsp70, Interleukin (IL -10, IL-6 and Tumor Necrosis Factor (TNF alpha concentrations were determined in 90 hospitalised geriatric patients (aged 83 ± 6 years and in 200 community-dwelling control subjects (100 elderly, aged 74 ± 5 years, and 100 young, aged 23 ± 3 years. In the community-dwelling elderly, serum Hsp70 and IL-10 concentrations were significantly lower and IL-6 was significantly higher when compared to healthy young control subjects. Elderly patients presenting inflammation (CRP serum levels ≥5 mg/L showed significantly (p = 0.007 higher Hsp70 values; and Hsp70 correlated positively (p Conclusions The present data provide new evidence that serum concentration of Hsp70 decreases with age in a normal population. Our study also shows that higher levels of Hsp70 are associated with inflammation and frailty in elderly patients.

  3. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  4. In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

    Energy Technology Data Exchange (ETDEWEB)

    Ebrahimi, Seyyed Mahmoud, E-mail: smebrahimi@shirazu.ac.ir [Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box 14155-3651,Tehran (Iran, Islamic Republic of); Research Center of Virus and Vaccine, Baqiyatallah University of Medical Science, P.O.Box 14155-3651, Tehran (Iran, Islamic Republic of); Dabaghian, Mehran [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of); Tebianian, Majid [Department of Biotechnology, Razi Vaccine and Serum Research Institute (RVSRI), P.O. Box 31975/148, Karaj, Tehran (Iran, Islamic Republic of); Zabeh Jazi, Mohammad Hossein [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of)

    2012-08-15

    Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS-PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0-33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

  5. In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

    International Nuclear Information System (INIS)

    Ebrahimi, Seyyed Mahmoud; Dabaghian, Mehran; Tebianian, Majid; Zabeh Jazi, Mohammad Hossein

    2012-01-01

    Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS–PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0–33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

  6. Oxygen glucose deprivation post-conditioning protects cortical neurons against oxygen-glucose deprivation injury: role of HSP70 and inhibition of apoptosis.

    Science.gov (United States)

    Zhao, Jian-hua; Meng, Xian-li; Zhang, Jian; Li, Yong-li; Li, Yue-juan; Fan, Zhe-ming

    2014-02-01

    In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (Pneuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.

  7. Deficiency of heat shock transcription factor 1 suppresses heat stress-associated increase in slow soleus muscle mass of mice.

    Science.gov (United States)

    Ohno, Y; Egawa, T; Yokoyama, S; Nakai, A; Sugiura, T; Ohira, Y; Yoshioka, T; Goto, K

    2015-12-01

    Effects of heat shock transcription factor 1 (HSF1) deficiency on heat stress-associated increase in slow soleus muscle mass of mice were investigated. Both HSF1-null and wild-type mice were randomly assigned to control and heat-stressed groups. Mice in heat-stressed group were exposed to heat stress (41 °C for 60 min) in an incubator without anaesthesia. Significant increase in wet and dry weights, and protein content of soleus muscle in wild-type mice was observed seven days after the application of the heat stress. However, heat stress had no impact on soleus muscle mass in HSF1-null mice. Neither type of mice exhibited much effect of heat stress on HSF mRNA expression (HSF1, HSF2 and HSF4). On the other hand, heat stress upregulated heat shock proteins (HSPs) at the mRNA (HSP72) and protein (HSP72 and HSP110) levels in wild-type mice, but not in HSF1-null mice. The population of Pax7-positive nuclei relative to total myonuclei of soleus muscle in wild-type mice was significantly increased by heat stress, but not in HSF1-null mice. Furthermore, the absence of HSF1 gene suppressed heat stress-associated phosphorylation of Akt and p70 S6 kinase (p-p70S6K) in soleus muscle. Heat stress-associated increase in skeletal muscle mass may be induced by HSF1 and/or HSF1-mediated stress response that activates muscle satellite cells and Akt/p70S6K signalling pathway. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  8. Beta-mangostin from Cratoxylum arborescens activates the intrinsic apoptosis pathway through reactive oxygen species with downregulation of the HSP70 gene in the HL60 cells associated with a G0/G1 cell-cycle arrest.

    Science.gov (United States)

    Omer, Fatima Abdelmutaal Ahmed; Hashim, Najihah Binti Mohd; Ibrahim, Mohamed Yousif; Dehghan, Firouzeh; Yahayu, Maizatulakmal; Karimian, Hamed; Salim, Landa Zeenelabdin Ali; Mohan, Syam

    2017-11-01

    Xanthones are phytochemical compounds found in a number of fruits and vegetables. Characteristically, they are noted to be made of diverse properties based on their biological, biochemical, and pharmacological actions. Accordingly, the apoptosis mechanisms induced by beta-mangostin, a xanthone compound isolated from Cratoxylum arborescens in the human promyelocytic leukemia cell line (HL60) in vitro, were examined in this study. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done to estimate the cytotoxicity effect of β-mangostin on the HL60 cell line. Acridine orange/propidium iodide and Hoechst 33342 dyes and Annexin V tests were conducted to detect the apoptosis features. Caspase-3 and caspase-9 activities; reactive oxygen species; real-time polymerase chain reaction for Bcl-2, Bax, caspase-3, and caspase-9 Hsp70 genes; and western blot for p53, cytochrome c, and pro- and cleavage-caspase-3 and caspase-9 were assessed to examine the apoptosis mechanism. Cell-cycle analysis conducted revealed that β-mangostin inhibited the growth of HL60 at 58 µM in 24 h. The administration of β-mangostin with HL60 caused cell morphological changes related to apoptosis which increased the number of early and late apoptotic cells. The β-mangostin-catalyzed apoptosis action through caspase-3, caspase-7, and caspase-9 activation overproduced reactive oxygen species which downregulated the expression of antiapoptotic genes Bcl-2 and HSP70. Conversely, the expression of the apoptotic genes Bax, caspase-3, and caspase-9 were upregulated. Meanwhile, at the protein level, β-mangostin activated the formation of cleaved caspase-3 and caspase-9 and also upregulated the p53. β-mangostin arrested the cell cycle at the G 0 /G 1 phase. Overall, the results for β-mangostin showed an antiproliferative effect in HL60 via stopping the cell cycle at the G 0 /G 1 phase and prompted the intrinsic apoptosis pathway.

  9. Targeting GRP75 improves HSP90 inhibitor efficacy by enhancing p53-mediated apoptosis in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Weiwei Guo

    Full Text Available Heat shock protein 90 (HSP90 inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70 family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.

  10. Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid.

    Directory of Open Access Journals (Sweden)

    Alexandra Silva

    Full Text Available Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90 chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.

  11. The Heat Shock Protein 26 Gene is Required for Ethanol Tolerance in Drosophila

    Directory of Open Access Journals (Sweden)

    Awoyemi A. Awofala

    2011-01-01

    Full Text Available Stress plays an important role in drug- and addiction-related behaviours. However, the mechanisms underlying these behavioural responses are still poorly understood. In the light of recent reports that show consistent regulation of many genes encoding stress proteins including heat shock proteins following ethanol exposure in Drosophila , it was hypothesised that transition to alcohol dependence may involve the dysregulation of the circuits that mediate behavioural responses to stressors. Thus, behavioural genetic methodologies were used to investigate the role of the Drosophila hsp26 gene, a small heat shock protein coding gene which is induced in response to various stresses, in the development of rapid tolerance to ethanol sedation. Rapid tolerance was quantified as the percentage difference in the mean sedation times between the second and first ethanol exposure. Two independently isolated P-element mutations near the hsp26 gene eliminated the capacity for tolerance. In addition, RNAi-mediated functional knockdown of hsp26 expression in the glial cells and the whole nervous system also caused a defect in tolerance development. The rapid tolerance phenotype of the hsp26 mutants was rescued by the expression of the wild-type hsp26 gene in the nervous system. None of these manipulations of the hsp26 gene caused changes in the rate of ethanol absorption. Hsp26 genes are evolutionary conserved, thus the role of hsp26 in ethanol tolerance may present a new direction for research into alcohol dependency.

  12. Acute heat stress induces differential gene expressions in the testes of a broiler-type strain of Taiwan country chickens.

    Science.gov (United States)

    Wang, Shih-Han; Cheng, Chuen-Yu; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Huang, San-Yuan

    2015-01-01

    The expression of testicular genes following acute heat stress has been reported in layer-type roosters, but few similar studies have been conducted on broilers. This study investigated the effect of acute heat stress on the gene expression in the testes of a broiler-type strain of Taiwan country chickens. Roosters were subjected to acute heat stress (38°C) for 4 h, and then exposed to 25°C, with testes collected 0, 2, and 6 h after the cessation of heat stress, using non-heat-stressed roosters as controls (n = 3 roosters per group). The body temperature and respiratory rate increased significantly (pstress. The numbers of apoptotic cells increased 2 h after the acute heat stress (79 ± 7 vs. 322 ± 192, control vs. heat stress; pstressed chickens from those of the controls, including genes involved in the response to stimulus, protein metabolism, signal transduction, cell adhesion, transcription, and apoptosis. The mRNA expressions of upregulated genes, including HSP25, HSP90AA1, HSPA2, and LPAR2, and of downregulated genes, including CDH5, CTNNA3, EHF, CIRBP, SLA, and NTF3, were confirmed through quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, numerous transcripts in the testes exhibited distinct expressions between the heat-stressed broiler-type and layer-type chickens. We concluded that the transcriptional responses of testes to acute heat stress may differ between the broiler-type and layer-type roosters. Whether the differential expression patterns associate with the heat-tolerance in the strains require a further exploration.

  13. Effects of long-term heat stress and dietary restriction on the expression of genes of steroidogenic pathway and small heat-shock proteins in rat testicular tissue.

    Science.gov (United States)

    Bozkaya, F; Atli, M O; Guzeloglu, A; Kayis, S A; Yildirim, M E; Kurar, E; Yilmaz, R; Aydilek, N

    2017-08-01

    The aim was to investigate the effects of long-term heat stress and dietary restriction on the expression of certain genes involving in steroidogenic pathway and small heat-shock proteins (sHSPs) in rat testis. Sprague Dawley rats (n = 24) were equally divided into four groups. Group I and II were kept at an ambient temperature of 22°C, while Groups III and IV were reared at 38°C for 9 weeks. Feed was freely available for Group I and Group III, while Group II and Group IV were fed 60% of the diet consumed by their ad libitum counterparts. At the end of 9 weeks, testicles were collected under euthanasia. Total RNA was isolated from testis tissue samples. Expression profiles of the genes encoding androgen-binding protein, follicle-stimulating hormone receptor, androgen receptor, luteinising hormone receptor, steroidogenic acute regulatory protein (StAR), cyclooxygenase-2 and sHSP genes were assessed at mRNA levels using qPCR. Long-term heat stress decreased the expression of StAR and HspB10 genes while dietary restriction upregulated StAR gene expression. The results suggested that long-term heat stress negatively affected the expression of StAR and HspB10 genes and the dietary restriction was able to reverse negative effect of heat stress on the expression of StAR gene in rat testis. © 2016 Blackwell Verlag GmbH.

  14. Toxic Potential of Synthesized Graphene Zinc Oxide Nanocomposite in the Third Instar Larvae of Transgenic Drosophila melanogaster (hsp70-lacZBg9

    Directory of Open Access Journals (Sweden)

    Yasir Hasan Siddique

    2014-01-01

    Full Text Available In the present study the graphene zinc oxide nanocomposite (GZNC was synthesized, characterized, and evaluated for its toxic potential on third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZBg9. The synthesized GZNC was characterized by X-ray diffraction (XRD, Fourier transform infrared spectroscopy (FTIR, thermogravimetric analysis (TGA, scanning electron microscopy (SEM, and transmission electron microscopy (TEM. The GZNC in 0.1% dimethyl sulphoxide (DMSO was sonicated for 10 minutes and the final concentrations 0.033, 0.099, 0.199, and 3.996 μg/μL of diet were established. The third instar larvae were allowed to feed on it separately for 24 and 48 hr. The hsp70 expression was measured by o-nitrophenyl-β-D-galactopyranoside assay, tissue damage was measured by trypan blue exclusion test, and β-galactosidase activity was monitored by in situ histochemical β-galactosidase staining. Oxidative stress was monitored by performing lipid peroxidation assay and total protein estimation. Ethidium bromide/acridine orange staining was performed on midgut cells for apoptotic index and the comet assay was performed for the DNA damage. The results of the present study showed that the exposure of 0.199 and 3.996 μg/μL of GZNC was toxic for both 24 hr and 48 hr of exposure. The doses of 0.033 μg/μL and 0.099 of GZNC showed no toxic effects on its exposure to the third instar larvae for 24 hr as well as 48 hr of duration.

  15. Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

    Science.gov (United States)

    Ochoa, Gina H; Clark, Ying Mei; Matsumoto, Brian; Torres-Ruiz, Jose A; Robles, Laura J

    2002-02-01

    Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.

  16. Effects of heat stress on respiratory burst, oxidative damage and SERPINH1 (HSP47) mRNA expression in rainbow trout Oncorhynchus mykiss.

    Science.gov (United States)

    Wang, Yanni; Liu, Zhe; Li, Zhen; Shi, Haina; Kang, Yujun; Wang, Jianfu; Huang, Jinqiang; Jiang, Li

    2016-04-01

    For rainbow trout Oncorhynchus mykiss, high temperature is a major abiotic stress that limits its growth and productivity. In this study, spleen macrophage respiratory burst (RB), serum superoxide dismutase (SOD), serum malondialdehyde (MDA) and mRNA expression of the SERPINH1 (HSP47) gene in different tissues (liver, spleen, head kidney and heart) were measured in unstressed (18 °C) and heat-stressed (25 °C) fish. Spleen macrophage RB activity, serum SOD activity and MDA content all increased significantly (P mykiss. In practice, close attention should be given to temperature changes in O. mykiss production to reduce the effects of high temperature.

  17. Effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 expression in juvenile chinese shrimp, Fenneropenaeus chinensis

    Science.gov (United States)

    Ding, Sen; Wang, Fang; Sun, Hao; Guo, Biao; Dong, Shuanglin

    2009-03-01

    Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively, decreased abruptly to salinity 24, lasted for another 2 d, and then was raised to its initial value 28. This was a complete salinity fluctuation cycle that afterwards repeated itself. After 32 days, the osmolarity in treatments D2, D4, D6 and D8 was significantly lower than that in treatment D0 ( P<0.05). There were significant differences in both muscle and eyestalks HSP70 expression among groups. The HSP70 expressions in muscle and eyestalks in group D4 were 61.4% and 57.0% higher, respectively, than that in the control group D0 ( P<0.05). There were, however, no significant differences in gill or hepatopancreas Na+-K+-ATPase activity between the treatments and the control.

  18. Biomarkers of dissolved oxygen stress in oysters: a tool for restoration and management efforts.

    Science.gov (United States)

    Patterson, Heather K; Boettcher, Anne; Carmichael, Ruth H

    2014-01-01

    The frequency and intensity of anoxic and hypoxic events are increasing worldwide, creating stress on the organisms that inhabit affected waters. To understand the effects of low dissolved oxygen stress on oysters, hatchery-reared oysters were placed in cages and deployed along with continuously recording environmental data sondes at a reef site in Mobile Bay, AL that typically experiences low oxygen conditions. To detect and measure sublethal stress, we measured growth and survival of oysters as well as expression of three biomarkers, heat shock protein 70 (HSP70), hypoxia inducible factor (HIF) and phospho-p38 MAP kinase, in tissues from juvenile and adult oysters. Survival rates were high for both juvenile and adult oysters. Expression levels of each of the 3 isoforms of HSP 70 were negatively correlated to dissolved oxygen (DO) concentrations, suggesting that HSP 70 is useful to quantify sublethal effects of DO stress. Results for HIF and phospho-p38 MAP kinase were inconclusive. Test deployments of oysters to assess expression of HSP 70 relative to environmental conditions will be useful, in addition to measuring abiotic factors, to identify appropriate sites for restoration, particularly to capture negative effects of habitat quality on biota before lethal impacts are incurred.

  19. Altered Cross-linking of HSP27 by Zerumbone as a Novel Strategy for Overcoming HSP27- mediated Resistance

    International Nuclear Information System (INIS)

    Choi, Seo Hyun; Lee, Yoon Jin; Lee, Hae June; Lee, Yun Sil; Kim, Joon; Seo, Woo Duck; Nam, Joo Won; Lee, Yoo Jin; Seo, Eun Kyung

    2010-01-01

    HSPs have diverse roles in the regulation of signal transduction and in numerous aspects of cell growth and death. Indeed, HSP90, HSP70, and HSP27 have each been implicated in promoting cancer. Most HSP27 exists as large oligomeric complexes ranging from 100- 800 kDa, which are probably stabilized by complex interactions between dimeric building blocks. The functional properties of HSP27 are dependent on the quaternary structure of the protein. For example, HSP27 acts as a chaperone and binds to cytochrome c or Daxx as a dimer. Therefore, the oligomerization pattern of HPS27 is believed to have HSP27-mediated protective functions. In this study, zerumbone (ZER), the cytotoxic component isolated from Zingiber zerumbet Smith, induced cross-linking of HSP27 protein by its insertion between the disulfide bond of HSP27, and ZERmediated altered cross-linking of HSP27 modified normal HSP27 dimerization, which resulted in a sensitizing effect to tumors after treatment with radiation. Therefore, altered cross-linking by ZER may be a novel strategy for inhibition of HSP27-mediated resistance

  20. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows*

    Science.gov (United States)

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-01-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (Pheat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat stress in dairy cows in the future. PMID:26055916

  1. Transport stress induces heart damage in newly hatched chicks via blocking the cytoprotective heat shock response and augmenting nitric oxide production.

    Science.gov (United States)

    Sun, F; Zuo, Y-Z; Ge, J; Xia, J; Li, X-N; Lin, J; Zhang, C; Xu, H-L; Li, J-L

    2018-04-20

    Transport stress affects the animal's metabolism and psychological state. As a pro-survival pathway, the heat shock response (HSR) protects healthy cells from stressors. However, it is unclear whether the HSR plays a role in transport stress-induced heart damage. To evaluate the effects of transport stress on heart damage and HSR protection, newly hatched chicks were treated with transport stress for 2 h, 4 h and 8 h. Transport stress caused decreases in body weight and increases in serum creatine kinase (CK) activity, nitric oxide (NO) content in heart tissue, cardiac nitric oxide syntheses (NOS) activity and NOS isoforms transcription. The mRNA expression of heat shock factors (HSFs, including HSF1-3) and heat shock proteins (HSPs, including HSP25, HSP40, HSP47, HSP60, HSP70, HSP90 and HSP110) in the heart of 2 h transport-treated chicks was upregulated. After 8 h of transport stress in chicks, the transcription levels of the same HSPs and HSF2 were reduced in the heart. It was also found that the changes in the HSP60, HSP70 and HSP90 protein levels had similar tendencies. These results suggested that transport stress augmented NO generation through enhancing the activity of NOS and the transcription of NOS isoforms. Therefore, this study provides new evidence that transport stress induces heart damage in the newly hatched chicks by blocking the cytoprotective HSR and augmenting NO production.

  2. A fraction from Petiveria alliacea induces apoptosis via a mitochondria-dependent pathway and regulates HSP70 expression

    OpenAIRE

    Susana Fiorentino; Diana Mercedes Castañeda; Claudia Patricia Urueña; Maria Claudia Cifuentes

    2009-01-01

    To evaluate the biological activity of Petiveria alliacea extracts on tumoral cells in vitro. Materials and methods. P.alliaceafractions prepared by a bioguided purification protocol were characterized by their biological activities on two human tumoral cell lines.Morphological changes, cell viability, mitochondrial membrane depolarization, nuclear staining and activity on HSP70 were analyzed.Results. The present study demonstrates that P.alliacea fractions can induce apoptosis in a mitochond...

  3. The 'tubulin-like' S1 protein of Spirochaeta is a member of the hsp65 stress protein family

    Science.gov (United States)

    Munson, D.; Obar, R.; Tzertzinis, G.; Margulis, L.

    1993-01-01

    A 65-kDa protein (called S1) from Spirochaeta bajacaliforniensis was identified as 'tubulin-like' because it cross-reacted with at least four different antisera raised against tubulin and was isolated, with a co-polymerizing 45-kDa protein, by warm-cold cycling procedures used to purify tubulin from mammalian brain. Furthermore, at least three genera of non-cultivable symbiotic spirochetes (Pillotina, Diplocalyx, and Hollandina) that contain conspicuous 24-nm cytoplasmic tubules displayed a strong fluorescence in situ when treated with polyclonal antisera raised against tubulin. Here we summarize results that lead to the conclusion that this 65-kDa protein has no homology to tubulin. S1 is an hsp65 stress protein homologue. Hsp65 is a highly immunogenic family of hsp60 proteins which includes the 65-kDa antigens of Mycobacterium tuberculosis (an active component of Freund's complete adjuvant), Borrelia, Treponema, Chlamydia, Legionella, and Salmonella. The hsp60s, also known as chaperonins, include E. coli GroEL, mitochondrial and chloroplast chaperonins, the pea aphid 'symbionin' and many other proteins involved in protein folding and the stress response.

  4. Molecular characterization of three Hsp90 from Pieris and expression patterns in response to cold and thermal stress in summer and winter diapause of Pieris melete.

    Science.gov (United States)

    Wu, Yue-Kun; Zou, Chao; Fu, Dao-Meng; Zhang, Wan-Na; Xiao, Hai-Jun

    2018-04-01

    Heat shock proteins (Hsps) have been linked to stresses and winter diapause in insects, but whether they are components of summer diapause is still unknown. In this study, complementary DNAs of Hsp90 from Pieris melete, Pieris rapae and Pieris canidia named PmHsp90, PrHsp90 and PcHsp90, respectively, were cloned and sequenced. The deduced amino acid sequence consisted of 718 amino acid residues with a putative molecular mass of 82.6, 82.6 and 82.7 kDa, respectively. The amino acid sequences contained all of the five conserved signature motifs in the Hsp90 family and a bHLH protein folding activity region. The differential expression pattern of PmHsp90 in response to summer diapause and winter diapause, which are related to heat/cold stress, was investigated. Cold stress induced Hsp90 up-regulation in summer and winter diapause pupae, but not in non-diapause individuals. Heat shock up-regulated PmHsp90 gradually with an increase in temperature in summer diapause, and PmHsp90 was rapidly up-regulated in winter diapause. After 30 min heat shock at 39°C, substantial up-regulation of PmHsp90 transcript levels were observed both in summer and winter diapause. However, in non-diapause a relatively stable expression was found under different durations of 39°C heat shock. Compared to the optimal treatment of 18°C for diapause development, a high temperature acclimation of 31°C induced PmHsp90 up-regulation in summer diapause, whereas a low temperature acclimation of 4°C induced up-regulation in winter diapause. The current results indicate that Hsp90 may play an important role in response to heat/cold stress both in summer and winter diapause. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  5. Characterization of small HSPs from Anemonia viridis reveals insights into molecular evolution of alpha crystallin genes among cnidarians.

    Directory of Open Access Journals (Sweden)

    Aldo Nicosia

    Full Text Available Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive study of the molecular evolution of α-crystallin gene family among cnidarians. Most of the H. magnipapillata sHSP genes share the same gene organization described for AvHSP28.6 and AvHSP27, differing from the sHSP genes of N. vectensis which mainly show an intronless architecture. The different genomic organization of sHSPs, the phylogenetic analyses based on protein sequences, and the relationships among Cnidarians, suggest that the A.viridis sHSPs represent the common ancestor from which H. magnipapillata genes directly evolved through segmental genome duplication. Additionally retroposition events may be considered responsible for the divergence of sHSP genes of N. vectensis from A. viridis. Analyses of transcriptional expression profile showed that AvHSP28.6 was constitutively expressed among different tissues from both ectodermal and endodermal layers of the adult sea anemones, under normal physiological conditions and also under different stress condition. Specifically, we profiled the transcriptional activation of AvHSP28.6 after challenges with different abiotic/biotic stresses showing induction by extreme temperatures, heavy metals exposure and immune stimulation. Conversely, no AvHSP27 transcript was detected in such dissected tissues, in adult whole body cDNA library or under stress conditions. Hence, the involvement of AvHSP28.6 gene in the sea anemone defensome is strongly suggested.

  6. Characterization of small HSPs from Anemonia viridis reveals insights into molecular evolution of alpha crystallin genes among cnidarians.

    Science.gov (United States)

    Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela; Costa, Salvatore

    2014-01-01

    Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain) are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive study of the molecular evolution of α-crystallin gene family among cnidarians. Most of the H. magnipapillata sHSP genes share the same gene organization described for AvHSP28.6 and AvHSP27, differing from the sHSP genes of N. vectensis which mainly show an intronless architecture. The different genomic organization of sHSPs, the phylogenetic analyses based on protein sequences, and the relationships among Cnidarians, suggest that the A.viridis sHSPs represent the common ancestor from which H. magnipapillata genes directly evolved through segmental genome duplication. Additionally retroposition events may be considered responsible for the divergence of sHSP genes of N. vectensis from A. viridis. Analyses of transcriptional expression profile showed that AvHSP28.6 was constitutively expressed among different tissues from both ectodermal and endodermal layers of the adult sea anemones, under normal physiological conditions and also under different stress condition. Specifically, we profiled the transcriptional activation of AvHSP28.6 after challenges with different abiotic/biotic stresses showing induction by extreme temperatures, heavy metals exposure and immune stimulation. Conversely, no AvHSP27 transcript was detected in such dissected tissues, in adult whole body cDNA library or under stress conditions. Hence, the involvement of AvHSP28.6 gene in the sea anemone defensome is strongly suggested.

  7. Small changes in gene expression of targeted osmoregulatory genes when exposing marine and freshwater threespine stickleback (Gasterosteus aculeatus to abrupt salinity transfers.

    Directory of Open Access Journals (Sweden)

    Annette Taugbøl

    Full Text Available Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13, cystic fibrosis transmembrane regulator (CFTR and a voltage gated potassium channel gene (KCNH4 and one stress related heat shock protein gene (HSP70 in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.

  8. Small changes in gene expression of targeted osmoregulatory genes when exposing marine and freshwater threespine stickleback (Gasterosteus aculeatus) to abrupt salinity transfers.

    Science.gov (United States)

    Taugbøl, Annette; Arntsen, Tina; Ostbye, Kjartan; Vøllestad, Leif Asbjørn

    2014-01-01

    Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.

  9. Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production.

    Science.gov (United States)

    Chung, Eun Joo; Jeong, Young-Il; Lee, Myoung-Ro; Kim, Yu Jung; Lee, Sang-Eun; Cho, Shin-Hyeong; Lee, Won-Ja; Park, Mi-Yeoun; Ju, Jung-Won

    2017-03-01

    Heat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90). rCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA. Both proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1β, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90. This study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective

  10. Hsp90: A New Player in DNA Repair?

    Directory of Open Access Journals (Sweden)

    Rosa Pennisi

    2015-10-01

    Full Text Available Heat shock protein 90 (Hsp90 is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis, transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR pathways that include: (i cell cycle arrest; (ii transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

  11. Attenuating brain edema, hippocampal oxidative stress, and cognitive dysfunction in rats using hyperbaric oxygen preconditioning during simulated high-altitude exposure.

    Science.gov (United States)

    Lin, Hung; Chang, Ching-Ping; Lin, Hung-Jung; Lin, Mao-Tsun; Tsai, Cheng-Chia

    2012-05-01

    We assessed whether hyperbaric oxygen preconditioning (HBO2P) in rats induced heat shock protein (HSP)-70 and whether HSP-70 antibody (Ab) preconditioning attenuates high altitude exposure (HAE)-induced brain edema, hippocampal oxidative stress, and cognitive dysfunction. Rats were randomly divided into five groups: the non-HBO2P + non-HAE group, the HBO2P + non-HAE group, the non-HBO2P + HAE group, the HBO2P + HAE group, and the HBO2P + HSP-70 Abs + HAE group. The HBO2P groups were given 100% O2 at 2.0 absolute atmospheres for 1 hour per day for 5 consecutive days. The HAE groups were exposed to simulated HAE (9.7% O2 at 0.47 absolute atmospheres of 6,000 m) in a hypobaric chamber for 3 days. Polyclonal rabbit anti-mouse HSP-70-neutralizing Abs were intravenously injected 24 hours before the HAE experiments. Immediately after returning to normal atmosphere, the rats were given cognitive performance tests, overdosed with a general anesthetic, and then their brains were excised en bloc for water content measurements and biochemical evaluation and analysis. Non-HBO2P group rats displayed cognitive deficits, brain edema, and hippocampal oxidative stress (evidenced by increased toxic oxidizing radicals [e.g., nitric oxide metabolites and hydroxyl radicals], increased pro-oxidant enzymes [e.g., malondialdehyde and oxidized glutathione] but decreased antioxidant enzymes [e.g., reduced glutathione, glutathione peroxide, glutathione reductase, and superoxide dismutase]) in HAE. HBO2P induced HSP-70 overexpression in the hippocampus and significantly attenuated HAE-induced brain edema, cognitive deficits, and hippocampal oxidative stress. The beneficial effects of HBO2P were significantly reduced by HSP-70 Ab preconditioning. Our results suggest that high-altitude cerebral edema, cognitive deficit, and hippocampal oxidative stress can be prevented by HSP-70-mediated HBO2P in rats.

  12. Caffeine Induces the Stress Response and Up-Regulates Heat Shock Proteins in Caenorhabditis elegans.

    Science.gov (United States)

    Al-Amin, Mohammad; Kawasaki, Ichiro; Gong, Joomi; Shim, Yhong-Hee

    2016-02-01

    Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

  13. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    2007-03-22

    Mar 22, 2007 ... Keywords. Caspases; Hsp90; Hsp70; Hsp60; Hsp27; hsr-omega. Abstract. Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper ...

  14. Molecular mechanisms of canalization: Hsp90 and beyond

    Indian Academy of Sciences (India)

    2006-03-26

    Mar 26, 2006 ... ... and regulatory circuits, accounting for the important role Hsp90 plays in ... by environmental stress, genetic or pharmaceutical targeting of Hsp90. The ... Here, we discuss the role of Hsp90 in canalization and organismal ...

  15. Examination the expression pattern of HSP70 heat shock protein in chicken PGCs and developing genital ridge

    OpenAIRE

    Mahek Anand; Roland Tóth; Alayu Kidane; Alexandra Nagy; Bence Lázár; Eszter Patakiné Várkonyi; Krisztina Liptói; Elen Gocza

    2016-01-01

    Chicken Primordial Germ cells (PGCs) are emerging pioneers in the field of applied embryology and stem cell technology. Now-a-days transgenic chickens are promising models to study human disease pathophysiology and drug designing. However, most of the molecular mechanism, which govern the stemness and pluripotency of chicken PGCs, not known in details. Recent studies have indicated the role of HSP70 in early embryonic development in many vertebrate species. Exposure of chicken to heat...

  16. The long-term effects of a life-prolonging heat treatment on the Drosophila melanogaster transcriptome suggest that heat shock proteins extend lifespan

    DEFF Research Database (Denmark)

    Sarup, Pernille Merete; Sørensen, Peter; Loeschcke, Volker

    2014-01-01

    Heat-induced hormesis, i.e. the beneficial effect of mild heat-induced stress, increases the average lifespan of many organisms. This effect, which depends on the heat shock factor, decreases the log mortality rate weeks after the stress has ceased. To identify candidate genes that mediate......-treated flies. Several hsp70 probe sets were up-regulated 1.7–2-fold in the mildly stressed flies weeks after the last heat treatment (P shock protein, Hsp70, is reported to return to normal levels of expression shortly after heat stress. We...... conclude that the heat shock response, and Hsp70 in particular, may be central to the heat-induced increase in the average lifespan in flies that are exposed to mild heat stress early in life....

  17. Urine heat shock protein 70 levels as a marker of urinary tract infection in children.

    Science.gov (United States)

    Yilmaz, Alev; Yildirim, Zeynep Yuruk; Emre, Sevinc; Gedikbasi, Asuman; Yildirim, Tarik; Dirican, Ahmet; Ucar, Evren Onay

    2016-09-01

    Heat shock proteins (HSPs) are a multi-family group of proteins which are upregulated by the cell in response to exposure to hazardous (stress) factors, including infectious agents, to prevent changes in protein structure. The aim of our study was to assess whether urine levels of the 70-kDa family of HSPs (HSP70s) increase in children with urinary tract infection (UTI) and to determine the optimal urine (u) HSP70 cut-off level to predict UTI in children. Forty patients with symptomatic UTI (UTI group), 30 healthy children (control group), 21 asymptomatic patients with proven bacterial contamination in their urine culture (contamination group) and 30 patients with fever caused by other infections (non-UTI infection group) were enrolled in the study. Random urine samples were obtained for measurement of HSP70 and creatinine (Cr) from all groups. Urine was collected prior to the treatment of UTI at the time of presentation and after treatment. Urine HSP70 levels were measured by enzyme-linked immunosorbent analysis. A dimercaptosuccinic acid (DMSA) scan was performed at 5-7 days after presentation in UTI group to distinguish patients with acute pyelonephritis from those with cystitis; based on this scan, no patients had acute pyelonephritis. Patients were classified with pyelonephritis in the presence of all of the following signs: axillary fever of ≥39 °C, leukocytosis and positivity for C-reactive protein. The mean urine HSP70:Cr ratio (uHSP70/Cr) prior to treatment was significantly higher in the UTI group (449.86 ± 194.33 pg/mg) than in the control, contamination and non-UTI infection groups (39.93 ± 47.61, 32.43 ± 9.09 and 45.14 ± 19.76, respectively; p = 0.0001). Using a cut-off of 158 pg/mg uHSP70/Cr for the prediction of UTI, the sensitivity and specificity of the assay were 100 and 100 %, respectively (area under the time-concentration curve = 1). The uHSP70/Cr was highest in the patients with clinical pyelonephritis (p

  18. Interactive effects of nutrition, reproductive state and pollution on molecular stress responses of mussels, Mytilus galloprovincialis Lamarck, 1819.

    Science.gov (United States)

    González-Fernández, Carmen; Albentosa, Marina; Sokolova, Inna

    2017-10-01

    Marine bivalves including mussels Mytilus galloprovincialis are commonly used as sentinels for pollution monitoring and ecosystem health assessment in the coastal zones. Use of biomarkers to assess the pollution effects assumes that the effects of pollutants on the biomarkers exceed the natural background variability; yet this assumption has rarely been tested. We exposed mussels at different reproductive stages and nutritive states to two concentrations of a polycyclic aromatic hydrocarbon (fluoranthene, 3 and 60 μg L -1 ) for three weeks. Expression levels of the molecular biomarkers related to the detoxification and general stress response [cytochrome P450 oxidase (CYP450), glutathione S-transferases (GST-α; GST-S1; GST-S2), the multixenobiotic resistance protein P-glycoprotein (PgP), metallothioneins (MT10 and MT20), heat shock proteins (HSP22, HSP70-2; HSP70-3; HSP70-4), as well as mRNA expression of two reproduction-related genes, vitellogenin (Vitel) and vitelline coat lysin M7 (VCLM7)] were measured. The mussels' nutrition and reproductive state affected the baseline mRNA levels of molecular biomarkers and modulated the transcriptional responses of biomarker genes to the pollutant exposure. Thus, mussel physiological state could act as a confounding factor in the evaluation of the response of pollution through molecular biomarkers. The biomarker baseline levels must be determined across a range of physiological states to enable the use of biomarkers in monitoring programs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Food availability is expressed through physiological stress indicators in nestling white ibis: A food supplementation experiment

    Science.gov (United States)

    Herring, G.; Cook, Mark I.; Gawlik, D.E.; Call, Erynn M.

    2011-01-01

    Physiological responses to environmental stress such as adrenocortical hormones and cellular stress proteins have recently emerged as potentially powerful tools for investigating physiological effects of avian food limitation. However, little is known about the physiological stress responses of free-living nestling birds to environmental variation in food availability. We experimentally tested how hydrologically mediated changes in food availability affect the physiological stress responses of juvenile white ibises Eudocimus albus in a fluctuating wetland. We provided supplementary food to free-living nestlings during 2years with contrasting hydrologic and food availability conditions, and used plasma (PCORT) and faecal (FCORT) corticosterone and heat shock proteins (HSP60 and HSP70) from first-hatched (A-nestlings) and second-hatched (B-nestlings) to detect relatively short- to long-term responses to food limitation. Nestling physiological stress responses were relatively low in all treatments during the year with optimal food availability, but PCORT, FCORT and HSP60 levels increased during the poor food year. FCORT and HSP60 responses were clearly due to nutritional condition as elevated concentrations were evident primarily in control nestlings. Significant year by hatch order interactions for both FCORT and HSP60 revealed that these increases were largely incurred by B-nestlings. FCORT and HSP60 responses were also well developed early in neonatal development and remained elevated for the duration of the experiment suggesting a chronic stress response. PCORT and HSP70 were less informative stress responses. The nutritionally mediated increases in FCORT and HSP60 provide compelling evidence that white ibis nestlings can be physiologically affected by environmental food levels. FCORT and HSP60 are effective indicators of nutritional mediated stress for nestling white ibises and potentially for other species prone to capture or handling stress. ?? 2010 The Authors

  20. Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

    Directory of Open Access Journals (Sweden)

    Dongli Wan

    Full Text Available Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2 were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.