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Sample records for shock protein 70-1

  1. 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

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

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    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.

  3. Cognitive Function and Heat Shock Protein 70 in Children With Temporal Lobe Epilepsy.

    Science.gov (United States)

    Oraby, Azza M; Raouf, Ehab R Abdol; El-Saied, Mostafa M; Abou-Khadra, Maha K; Helal, Suzette I; Hashish, Adel F

    2017-01-01

    We conducted the present study to examine cognitive function and serum heat shock protein 70 levels among children with temporal lobe epilepsy. The Stanford-Binet Intelligence Test was carried out to examine cognitive function in 30 children with temporal lobe epilepsy and 30 controls. Serum heat shock protein 70 levels were determined with an enzyme-linked immunosorbent assay. The epilepsy group had significantly lower cognitive function testing scores and significantly higher serum heat shock protein 70 levels than the control group; there were significant negative correlations between serum heat shock protein 70 levels and short-term memory and composite scores. Children with uncontrolled seizures had significantly lower verbal reasoning scores and significantly higher serum heat shock protein 70 levels than children with controlled seizures. Children with temporal lobe epilepsy have cognitive dysfunction and elevated levels of serum heat shock protein 70, which may be considered a stress biomarker.

  4. Role of heat shock protein 70 in innate alloimmunity

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    Walter G. eLand

    2012-01-01

    Full Text Available This article briefly describes our own experience with the proven demonstration of heat shock protein 70 in reperfused renal allografts from brain-deaddonors and reflects about its potential role as a typical damage-associated molecular pattern (DAMP in the setting of innate alloimmunity. In fact, our group was able to demonstrate a dramatic up-regulation of heat shock protein 70 expression after postischemic reperfusion of renal allografts. Of note, up-regulation of this stress protein expression, although to a lesser extent, was already observed after cold storage of the organ indicating that this molecule is already induced in the stressed organism of a brain-dead donor. However, whether or not the dramatic up-regulation of heat shock protein 70 expression contributes to mounting an innate alloimmune response cannot be judged in view of these clinical findings.Nevertheless, heat shock protein 70, since generated in association with postischemic reperfusion-induced allograft injury, can be called a typical DAMP - as can everymolecule be termed a DAMP that is generated in associationwith any stressful tissue injury regardless of its final positive or negative regulatory function within the innate immune response elicited by it.In fact, as we discuss in this article, the context-dependent, even contradistinctive activities of heat shock protein 70 reflect the biological phenomenon that, throughout evolution, mammals have developed an elaborate network of positive and negative regulatory mechanisms, which provide balance between defensive and protective measures against unwarranted destruction of the host. In this sense, up-regulated expression of heat shock protein 70 in an injured allograft might reflect a pure protective response against the severe oxidative injury of a reperfused donor organ. On the other hand, up-regulated expression of this stress protein in an injured allograft might reflect a(futile attempt of the innate immune system to

  5. The pretective effects of heat shock protein 70 on radiation injury of V79 cells

    International Nuclear Information System (INIS)

    Qin Yongchun; Zhang Baoguo; Hong Chengjiao

    2008-01-01

    Westem blot was used to detect the expression of heat shock protein 70 in V79 cells after heat shock pretreatment; V79 cells were irradiated using γ-ray after heat shock pretreatment, survival rate was observed using Colony formation assay. Our study shows that 1) the overexpression of heat shock protein 70 was observed in cells recovering for 1 hour after heat shock pretreatment, with peak expression in cells recovering for 4 hours, and could last for 24 hours; 2) heat shock pretreatment was able to elevate survival rate of V79 cells after irradiation by 60 Co γ ray (when the irradiation dose was less than 6 Gy). The results indicate that heat shock protein 70 has protective effect on radiation induced cell death of V79 cells (when the irradiation dose was less than 6 Gy). (authors)

  6. Increased expression of heat shock protein 70 and heat shock factor 1 in chronic dermal ulcer tissues treated with laser-aided therapy.

    Science.gov (United States)

    Zhou, Jian-da; Luo, Cheng-qun; Xie, Hui-qing; Nie, Xin-min; Zhao, Yan-zhong; Wang, Shao-hua; Xu, Yi; Pokharel, Pashupati Babu; Xu, Dan

    2008-07-20

    Chronic dermal ulcers are also referred to as refractory ulcers. This study was conducted to elucidate the therapeutic effect of laser on chronic dermal ulcers and the induced expression of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70) in wound tissues. Sixty patients with 84 chronic dermal ulcers were randomly divided into traditional therapy and laser therapy groups. Laser treatment was performed in addition to traditional therapy in the laser therapy group. The treatment efficacy was evaluated after three weeks. Five tissue sections of healing wounds were randomly collected along with five normal skin sections as controls. HSP70-positive cells from HSP70 immunohistochemical staining were counted and the gray scale of positive cells was measured for statistical analysis. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the mRNA and protein expressions of HSF1 and HSP70. The cure rate of the wounds and the total efficacy in the laser therapy group were significantly higher than those in the traditional therapy group (P ulcers plays a facilitating role in healing due to the mechanism of laser-activated endogenous heat shock protection in cells in wound surfaces.

  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

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    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. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  9. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

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    Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

    1996-03-19

    Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

  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. Plasma autoantibodies against heat shock protein 70, enolase 1 and ribonuclease/angiogenin inhibitor 1 as potential biomarkers for cholangiocarcinoma.

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    Rucksak Rucksaken

    Full Text Available The diagnosis of cholangiocarcinoma (CCA is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1 and CCA cell lines (M055, M214 and M139 were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70, enolase 1 (ENO1 and ribonuclease/angiogenin inhibitor 1 (RNH1 obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity. Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001. In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05. Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.

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

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    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.

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

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    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...

  14. 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.

  15. 14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Liu, Chia-Chia; Wang, John; Shyue, Song-Kun; Sung, Li-Ying; Liou, Jun-Yang; Jan, Yee-Jee; Ko, Bor-Sheng; Wu, Yao-Ming; Liang, Shu-Man; Chen, Shyh-Chang; Lee, Yen-Ming; Liu, Tzu-An; Chang, Tzu-Ching

    2014-01-01

    14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear. We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay. In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β. Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC

  16. 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

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    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.

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

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    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.

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

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    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

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

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    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.

  20. 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...

  1. 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.

  2. Salivary protein histatin 3 regulates cell proliferation by enhancing p27{sup Kip1} and heat shock cognate protein 70 ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Imamura, Yasuhiro, E-mail: yimamura@po.mdu.ac.jp [Department of Pharmacology, Matsumoto Dental University, Shiojiri, Nagano 399-0781 (Japan); Wang, Pao-Li [Department of Bacteriology, Osaka Dental University, Hirakata, Osaka 573-1121 (Japan); Masuno, Kazuya [Department of Dental Education Innovation, Osaka Dental University, Hirakata, Osaka 573-1121 (Japan); Sogawa, Norio [Department of Pharmacology, Matsumoto Dental University, Shiojiri, Nagano 399-0781 (Japan)

    2016-02-05

    Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5–8 was essential for enhancing p27{sup Kip1} (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27{sup Kip1} and HSC70 during the G{sub 1}/S transition of HGFs as opposed to histatin 3-M(5–8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5–8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination, whereas histatin 3-M(5–8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G{sub 1}/S transition, via the ubiquitin–proteasome system of p27{sup Kip1} and HSC70. - Highlights: • KRHH amino acid sequence was required in histatin 3 to bind HSC70. • Histatin 3 enhanced HSC70 binding to p27{sup Kip1} during the G{sub 1}/S transition in HGFs. • KRHH sequence stimulated DNA synthesis and promoted cell survival. • Histatin 3 dose-dependently enhanced both p27{sup Kip1} and HSC70 ubiquitination. • Histatin 3 stimulates cell proliferation via the ubiquitin–proteasome system.

  3. 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.

  4. 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.

  5. 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

  6. Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice.

    Science.gov (United States)

    Shen, Hai-Ying; He, Jin-Cai; Wang, Yumei; Huang, Qing-Yuan; Chen, Jiang-Fan

    2005-12-02

    As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.

  7. 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.

  8. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    Science.gov (United States)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  9. 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,

  10. Molecular cloning and expression of a heat-shock cognate 70 (hsc70) gene from swordtail fish ( Xiphophorus helleri)

    Science.gov (United States)

    Li, Ningqiu; Fu, Xiaozhe; Han, Jingang; Shi, Cunbin; Huang, Zhibin; Wu, Shuqin

    2013-07-01

    Heat shock proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In the present study, a full-length cDNA, encoding the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), was isolated from swordtail fish ( Xiphophorus helleri) and designated as XheHsc70. The Xhehsc70 cDNA was 2 104 bp long with an open reading frame of 1 941 bp, and it encoded a protein of 646 amino acids with a theoretical molecular weight of 70.77 kDa and an isoelectric point of 5.04. The deduced amino acid sequence shared 94.1%-98.6% identities with the Hsc70s from a number of other fish species. Tissue distribution results show that the Xhehsc70 mRNA was expressed in brain, heart, head kidney, kidney, spleen, liver, muscle, gill, and peripheral blood. After immunization with formalin-killed Vibrio alginolyticus cells there was a significant increase in the Xhehsc70 mRNA transcriptional level in the head kidney of the vaccinated fish compared with in the control at 6, 12, 24, and 48 h as shown by quantitative real time RT-PCR. Based on an analysis of the amino acid sequence of XheHsc70, its phylogeny, and Xhehsc70 mRNA expression, XheHsc70 was identified as a member of the cytoplasmic Hsc70 (constitutive) subfamily of the Hsp70 family of heat shock proteins, suggesting that it may play a role in the immune response. The Xhehsc70 cDNA sequence reported in this study was submitted to GenBank under the accession number JF739182.

  11. Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2.

    Science.gov (United States)

    Wang, Fengping; Qiu, Ye; Zhang, Huifang M; Hanson, Paul; Ye, Xin; Zhao, Guangze; Xie, Ronald; Tong, Lei; Yang, Decheng

    2017-07-01

    We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70-overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans-acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap-dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt-mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K- and cell division cycle protein 2 homolog (Cdc2)-mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection. © 2017 John Wiley & Sons Ltd.

  12. FLZ Attenuates α-Synuclein-Induced Neurotoxicity by Activating Heat Shock Protein 70.

    Science.gov (United States)

    Bao, Xiu-Qi; Wang, Xiao-Liang; Zhang, Dan

    2017-01-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. The pathology of PD is caused by progressive degeneration of dopaminergic neurons and is characterized by the presence of intracellular inclusions known as Lewy bodies, composed mainly of α-synuclein. Heat shock proteins (HSPs) are crucial in protein quality control in cells. HSP70 in particular prevents the aggregation of protein aggregation, such as α-synuclein, providing a degree of protection against PD. The compound FLZ has been shown to protect several PD models in previous studies and was reported as an HSP inducer to protect against MPP + -induced neurotoxicity, but the mechanism remains unclear. In this study, we investigated the effects of FLZ-mediated HSP70 induction in α-synuclein transgenic mice and cells. FLZ treatment alleviated motor dysfunction and improved dopaminergic neuronal function in α-synuclein transgenic mice. HSP70 protein expression and transcriptional activity were increased by FLZ treatment, eliciting a reduction of α-synuclein aggregation and associated toxicity. The inhibition of HSP70 by quercetin or HSP70 siRNA markedly attenuated the neuroprotective effects of FLZ, confirming that FLZ exerted a neuroprotective effect through HSP70. We revealed that FLZ directly bound to and increased the expression of Hip, a cochaperone of HSP70, which in turn enhanced HSP70 activity. In conclusion, we defined a critical role for HSP70 and its cochaperones activated by FLZ in preventing neurodegeneration and proposed that targeting the HSP70 system may represent a potential therapy for α-synuclein-related diseases, such as PD.

  13. 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...

  14. Modeling and Docking Studies on Novel Mutants (K71L and T204V of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2014-04-01

    Full Text Available The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO were mutated. 3D mutant models (K71L and T204V using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255, as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  17. 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

  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. Circulating Heat Shock Protein 70 in Health, Aging and Disease

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    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.

  20. Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

    Science.gov (United States)

    Taldone, Tony; Kang, Yanlong; Patel, Hardik J; Patel, Maulik R; Patel, Pallav D; Rodina, Anna; Patel, Yogita; Gozman, Alexander; Maharaj, Ronnie; Clement, Cristina C; Lu, Alvin; Young, Jason C; Chiosis, Gabriela

    2014-02-27

    The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

  1. 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.

  2. 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.

  3. The Synergistic Effects of Heat Shock Protein 70 and Ginsenoside Rg1 against Tert-Butyl Hydroperoxide Damage Model In Vitro

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    Dan Lu

    2015-01-01

    Full Text Available Neural stem cells (NSCs transplanted is one of the hottest research to treat Alzheimer’s disease (AD, but cholinergic neurons from stem cells were also susceptible to cell death which Heat shock protein 70 (HSP70 was affirmed to reverse. Related to cognitive impairment, cholinergic nervous cells should be investigated and ginsenoside Rg1 (G-Rg1 was considered to increase them. We chose tert-butyl hydroperoxide (t-BHP damage model to study in vitro. Functional properties of our recombination plasmid pEGFP-C2-HSP70 were affirmed by SH-SY5Y cells. To opposite the transitory appearance of HSP70, NSCs used as the vectors of HSP70 gene overexpressed HSP70 for at least 7 days in vitro. After transfection for 3 days, G-Rg1 pretreatment for 4 hours, and coculture for 3 days, the expression of acetylcholinesterase (ChAT, synaptophysin, and the ratio of NeuN and GFAP were assessed by western blot; Morphological properties were detected by 3D reconstruction and immunofluorescence. ChAT was markedly improved in the groups contained G-Rg1. In coculture system, the ratio of neurons/astrocytes and the filaments of neurons were increased; apoptosis cells were decreased, compared to monotherapy (P<0.05. In conclusion, we demonstrated that, as a safe cotreatment affirmed in vitro, overexpression of HSP70 in NSCs plus G-Rg1 promoted nervous cells regeneration from chronic oxidative damage.

  4. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  5. 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.

  6. 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.

  7. 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.

  8. Gene expression of heat shock protein 70, interleukin-1β and tumor necrosis factor α as tools to identify immunotoxic effects on Xenopus laevis: A dose–response study with benzo[a]pyrene and its degradation products

    International Nuclear Information System (INIS)

    Martini, Federica; Fernández, Carlos; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    The exposure to benzo[a]pyrene (B[a]P) results in an alteration of immune function in mammals and fish, and the analysis of cytokine mRNA levels has been suggested for predicting the immunomodulatory potential of chemicals. To obtain evidence of the innate immune responses to B[a]P in Xenopus laevis, the present study monitored the mRNA expression of interleukin 1-β (IL-1β), tumor necrosis factor α (TNF-α) and heat shock protein 70 (HSP70) in a laboratorial exposure. Tadpoles exposed to 8.36, 14.64, 89.06 and 309.47 μg/L of B[a]P,were used for detecting hsp70, IL-1β and TNF-α mRNA induction. A dose–response increase in the expression of hsp70 and IL-1β mRNA was found. The results of this study confirmed the use of hsp70 and IL-1β, but not TNF-α, as sensitive indicators of immunotoxic effect of B[a]P in X. laevis. Further research would be required for the validation of these endpoints. - Highlights: ► We study innate immune responses to benzo[a]pyrene in Xenopus laevis. ► mRNA expression of three typical proinflammatory proteins was monitored. ► Heat shock protein 70 mRNA induction showed a concentration/response/time relationship. ► Interleukin 1-β also showed a clear concentration/response relationship. ► Interleukin 1-β and heat shock protein 70 are useful indicators of immunotoxic effects. - The present study analyzed the use of cytokine mRNA levels as an earlier tool for predicting immunotoxicological risks to Xenopus laevis in a dose–response pattern.

  9. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

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    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  10. Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

    Science.gov (United States)

    Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

    2016-11-01

    Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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

  12. Induction of Heat Shock Protein 70 Ameliorates Ultraviolet-Induced Photokeratitis in Mice

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    Yukihiro Horie

    2013-01-01

    Full Text Available Acute ultraviolet (UV B exposure causes photokeratitis and induces apoptosis in corneal cells. Geranylgeranylacetone (GGA is an acyclic polyisoprenoid that induces expression of heat shock protein (HSP70, a soluble intracellular chaperone protein expressed in various tissues, protecting cells against stress conditions. We examined whether induction of HSP70 has therapeutic effects on UV-photokeratitis in mice. C57 BL/6 mice were divided into four groups, GGA-treated (500 mg/kg/mouse and UVB-exposed (400 mJ/cm2, GGA-untreated UVB-exposed (400 mJ/cm2, GGA-treated (500 mg/kg/mouse but not exposed and naive controls. Eyeballs were collected 24 h after irradiation, and corneas were stained with hematoxylin and eosin (H&E and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL. HSP70, reactive oxygen species (ROS production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and protein kinase B (Akt expression were also evaluated. Irradiated corneal epithelium was significantly thicker in the eyes of mice treated with GGA compared with those given the vehicle alone (p < 0.01. Significantly fewer TUNEL-positive cells were observed in the eyes of GGA-treated mice than controls after irradiation (p < 0.01. Corneal HSP70 levels were significantly elevated in corneas of mice treated with GGA (p < 0.05. ROS signal was not affected by GGA. NF-κB activation was reduced but phospho-(Ser/Ther Akt substrate expression was increased in corneas after irradiation when treated with GGA. GGA-treatment induced HSP70 expression and ameliorated UV-induced corneal damage through the reduced NF-κB activation and possibly increased Akt phosphorilation.

  13. 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

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

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    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.

  15. 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

  16. 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.

  17. 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

    Science.gov (United States)

    Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

    2013-01-01

    We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

  18. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

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    Fei Jiang

    Full Text Available Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu and heat shock protein 70 (HSP 70 are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70, IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

  19. 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.

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

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    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.

  1. 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.

  2. TARGETED DELETION OF INDUCIBLE HEAT SHOCK PROTEIN 70 ABROGATES THE LATE INFARCT-SPARING EFFECT OF MYOCARDIAL ISCHEMIC PRECONDITIONING

    Science.gov (United States)

    Abstract submitted for 82nd annual meeting of the American Association for Thoracic Surgery, May 4-8, 2002 in Washington D.C.Targeted Deletion of Inducible Heat Shock Protein 70 Abrogates the Late Infarct-Sparing Effect of Myocardial Ischemic PreconditioningCraig...

  3. 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.

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

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    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.

  5. 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.

  6. N,N'-dinitrosopiperazine--mediated heat-shock protein 70-2 expression is involved in metastasis of nasopharyngeal carcinoma.

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    Zhengke Peng

    Full Text Available N,N'-Dinitrosopiperazine (DNP is invovled in nasopharyngeal carcinoma (NPC development and metastasis, and it shows organ specificity to the nasopharyngeal epithelium. Herein, we demonstrate that DNP induces heat-shock protein (HSP 70-2 expression in NPC cells (6-10B at a non-cytotoxic concentration. DNP induced HSP70-2 expression in a dose- and time- dependent manner, but showed no effect on other HSP70 family members. Furthermore, DNP also increased HSP70-2 RNA transcription through directly binding to the hypoxia-responsive elements (HRE and heat shock elements (HSE located in the HSP70-2 promoter. DNP-mediated HSP70-2 expression might act through enhancing the transcription of HSP70-2 RNA. Importantly, DNP induced motility and invasion of 6-10B cells dose- and time-dependently, and DNP-mediated NPC metastasis was confirmed in nude mice, which showed high HSP70-2 expression in the metastatic tumor tissue. However, the motility and invasion of NPC cells that were stably transfected using short interfering RNA against HSP70-2 could not effectively induce DNP. These results indicate that DNP induces HSP70-2 expression through increasing HSP70-2 transcription, increases the motility and invasion of cells, and promotes NPC tumor metastasis. Therefore, DNP mediated HSP70-2 expression may be an important factor of NPC-high metastasis.

  7. 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

  8. P53 and heat shock protein 70 expressions in colorectal adenocarcinoma

    International Nuclear Information System (INIS)

    Shotar, Ali M.

    2005-01-01

    To examine the localization and over expression of heat shock protein 70 (HSP70) and p53 in patients with colorectal cancer and compared it with control tissue (including normal colon tissue). This was a retrospective study of 60 patients with colorectal adenocarcinoma at the Jordan University of Science and Technology (JUST), Irbid, Jordan from 1997 to 2000. The Pathology Department at JUST is the chief provider of surgical pathology services in the north of Jordan. It receives specimens from both government and private hospitals. Immunohistochemistry was the technique of choice. The HSP70 was over expressed more highly in colorectal cancers than in the control tissue. Immuno-histochemistry showed that over expression of HSP70 had no statistically significant difference with any of the different prognostic factors assessed, mainly the grade and the stage. The p53 was over expressed in 60% of the cases. Control tissue (normal colon) was negative, p53, cell-cycle-related oncogene product, was strongly over expressed in the nuclei of the cancer cells of the cancer tissue. We found no significant difference in terms of size, patient age, lymph node state, and stage. The rate of expression was significantly less in high grade tumors than in intermediate and low grade ones. The strong expression however, may be valuable in estimating a prognosis for patients with colo-rectal carcinoma. (author)

  9. Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

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    Go Sakai

    2017-11-01

    Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

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

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    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.

  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.

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    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. 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.

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

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

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    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/.

  15. 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

  16. 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.

  17. A family of related proteins is encoded by the major Drosophila heat shock gene family

    International Nuclear Information System (INIS)

    Wadsworth, S.C.

    1982-01-01

    At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites

  18. 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.

  19. 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

  20. Heat shock protein 90 in neurodegenerative diseases

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    Rodina Anna

    2010-06-01

    Full Text Available Abstract Hsp90 is a molecular chaperone with important roles in regulating pathogenic transformation. In addition to its well-characterized functions in malignancy, recent evidence from several laboratories suggests a role for Hsp90 in maintaining the functional stability of neuronal proteins of aberrant capacity, whether mutated or over-activated, allowing and sustaining the accumulation of toxic aggregates. In addition, Hsp90 regulates the activity of the transcription factor heat shock factor-1 (HSF-1, the master regulator of the heat shock response, mechanism that cells use for protection when exposed to conditions of stress. These biological functions therefore propose Hsp90 inhibition as a dual therapeutic modality in neurodegenerative diseases. First, by suppressing aberrant neuronal activity, Hsp90 inhibitors may ameliorate protein aggregation and its associated toxicity. Second, by activation of HSF-1 and the subsequent induction of heat shock proteins, such as Hsp70, Hsp90 inhibitors may redirect neuronal aggregate formation, and protect against protein toxicity. This mini-review will summarize our current knowledge on Hsp90 in neurodegeneration and will focus on the potential beneficial application of Hsp90 inhibitors in neurodegenerative diseases.

  1. Pilot study of intratumoral injection of recombinant heat shock protein 70 in the treatment of malignant brain tumors in children

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    Shevtsov MA

    2014-06-01

    Full Text Available Maxim A Shevtsov,1,2 Alexander V Kim,2 Konstantin A Samochernych,2 Irina V Romanova,3 Boris A Margulis,1 Irina V Guzhova,1 Igor V Yakovenko,2 Alexander M Ischenko,4 William A Khachatryan2 1Institute of Cytology of the Russian Academy of Sciences, 2AL Polenov Russian Research Scientific Institute of Neurosurgery, 3IM Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, 4Research Institute of Highly Pure Biopreparations, St Petersburg, Russian Federation Abstract: Intratumoral injections of recombinant heat shock protein (Hsp70 were explored for feasibility in patients with brain tumors. Patients aged 4.5–14 years with untreated newly diagnosed tumors (n=12 were enrolled. After tumor resection, five injections of recombinant Hsp70 (total 2.5 mg were administered into the resection cavity through a catheter. Before administration of Hsp70 and after the last injection, specific immune responses to the autologous tumor lysate were evaluated using the delayed-type hypersensitivity test. Further, peripheral blood was monitored to identify possible changes in lymphocyte subpopulations, cytokine levels, and the cytolytic activity of natural killer cells. The follow-up period in this trial was 12 months. Intratumoral injections of Hsp70 were well tolerated by patients. One patient had a complete clinical response documented by radiologic findings and one patient had a partial response. A positive delayed-type hypersensitivity test was observed in three patients. In peripheral blood, there was a shift from cytokines provided by Th2 cells toward cytokines of a Th1-cell-mediated response. These data corresponded to changes in lymphocyte subpopulations. Immunosuppressive T-regulatory cell levels were also reduced after injection of Hsp70, as well as production of interleukin-10. The cytolytic activity of natural killer cells was unchanged. The present study demonstrates the feasibility of intratumoral delivery

  2. 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.

  3. ATF1 Modulates the Heat Shock Response by Regulating the Stress-Inducible Heat Shock Factor 1 Transcription Complex

    Science.gov (United States)

    Takii, Ryosuke; Fujimoto, Mitsuaki; Tan, Ke; Takaki, Eiichi; Hayashida, Naoki; Nakato, Ryuichiro; Shirahige, Katsuhiko

    2014-01-01

    The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation. PMID:25312646

  4. 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.

  5. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Dufresne, Philippe J.; Thivierge, Karine; Cotton, Sophie; Beauchemin, Chantal; Ide, Christine; Ubalijoro, Eliane; Laliberte, Jean-Francois; Fortin, Marc G.

    2008-01-01

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

  6. 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.

  7. Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster.

    Science.gov (United States)

    Ahamed, Maqusood; Posgai, Ryan; Gorey, Timothy J; Nielsen, Mark; Hussain, Saber M; Rowe, John J

    2010-02-01

    Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 microg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity. Copyright 2009 Elsevier Inc. All rights reserved.

  8. Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Ahamed, Maqusood; Posgai, Ryan; Gorey, Timothy J.; Nielsen, Mark; Hussain, Saber M.; Rowe, John J.

    2010-01-01

    Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 μg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity.

  9. Circulating Heat Shock Proteins in Women With a History of Recurrent Vulvovaginitis

    Directory of Open Access Journals (Sweden)

    P. C. Giraldo

    1999-01-01

    70-kDa heat shock proteins (hsp60 and hsp70, respectively in the circulation of women with or without a history of recurrent BV or candidal vaginitis and with or without a current lower genital tract infection. Heat shock protein expression is associated with a down-regulation of proinflammatory immune responses that would inhibit microbial infection.

  10. Mast cell chymase degrades the alarmins heat shock protein 70, biglycan, HMGB1, and interleukin-33 (IL-33) and limits danger-induced inflammation.

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-03

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W(sash))-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.

  11. Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan, HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation*

    Science.gov (United States)

    Roy, Ananya; Ganesh, Goutham; Sippola, Helena; Bolin, Sara; Sawesi, Osama; Dagälv, Anders; Schlenner, Susan M.; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Kjellén, Lena; Hellman, Lars; Åbrink, Magnus

    2014-01-01

    During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(Wsash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation. PMID:24257755

  12. [Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

    Science.gov (United States)

    Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

    2015-10-01

    To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 m

  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. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells

    International Nuclear Information System (INIS)

    Santoro, M.G.; Garaci, E.; Amici, C.

    1989-01-01

    Prostaglandins (PGs)A 1 and J 2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA 1 and PGJ 2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ 2 treatment. p74 was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production

  15. Effect of Ramadan fasting on serum heat shock protein 70 and serum lipid profile.

    Science.gov (United States)

    Zare, A; Hajhashemi, M; Hassan, Z M; Zarrin, S; Pourpak, Z; Moin, M; Salarilak, S; Masudi, S; Shahabi, S

    2011-07-01

    Ramadan, the holy month for the Islamic world, is a period every year when food and fluid intake is restricted to the pre-sunrise and post-sunset hours. The aim of this study was to evaluate the effect of Ramadan fasting on the serum concentration of heat shock protein 70 (HSP70) and serum lipid profile in healthy men. A total of 32 male volunteers with a mean age of 28.5 (range 23-37) years were selected for the study. Blood samples were obtained one day prior to Ramadan and on the 3rd and 25th days of fasting. Serum HSP70, triglyceride (TG), cholesterol (Chol), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), LDL/HDL and Chol/HDL ratios were investigated. It was observed that the mean concentrations of serum HSP70 and HDL on the 25th day of Ramadan were significantly higher than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels on the 3rd day of Ramadan was significantly higher than those recorded one day before Ramadan. Mean concentrations of serum TG, Chol, LDL, and LDL/HDL and Chol/HDL ratios on the 25th day of Ramadan were significantly lower than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels found on the 3rd day of Ramadan were also significantly lower than those recorded one day before Ramadan. Ramadan fasting increases serum HSP70 and improves serum lipid profile.

  16. 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

  17. 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

  18. Heat shock cognate protein 70 contributes to Brucella invasion into trophoblast giant cells that cause infectious abortion

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    Furuoka Hidefumi

    2008-12-01

    Full Text Available Abstract Background The cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells. Results We observed internalization and intracellular growth of B. abortus in cultured TG cells. A monoclonal antibody that inhibits bacterial internalization was isolated and this reacted with heat shock cognate protein 70 (Hsc70. Depletion and over expression of Hsc70 in TG cells inhibited and promoted bacterial internalization, respectively. IFN-γ receptor was expressed in TG cells and IFN-γ treatment enhanced the uptake of bacteria by TG cells. Administering the anti-Hsc70 antibody to pregnant mice served to prevent infectious abortion. Conclusion B. abortus infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.

  19. 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.

  20. 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.

  1. 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.

  2. Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IκB/NF-κB cascade by facilitating IκB kinase renaturation and blocking its further denaturation

    International Nuclear Information System (INIS)

    Lee, Kyoung-Hee; Lee, Choon-Taek; Kim, Young Whan; Han, Sung Koo; Shim, Young-Soo; Yoo, Chul-Gyu

    2005-01-01

    Heat shock (HS) treatment has been previously shown to suppress the IκB/nuclear factor-κB (NF-κB) cascade by denaturing, and thus inactivating IκB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IκB/NF-κB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-α-induced activation of the IκB/NF-κB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-α-induced activation of the IκB/NF-κB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-α-induced IκBα degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IκBα stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IκB/NF-κB cascade by facilitating the renaturation of IKK and blocking its further denaturation

  3. Identification of an exported heat shock protein 70 in Plasmodium falciparum

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    Grover Manish

    2013-01-01

    Full Text Available Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer’s clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

  4. 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.

  5. Responses of heat shock protein 70 and caspase-3/7 to dietary selenomethionine in juvenile white sturgeon

    Directory of Open Access Journals (Sweden)

    Weifang Wang

    2016-03-01

    Full Text Available An 8-week feeding trial was conducted to investigate the responses of juvenile white sturgeon (Acipenser transmontanus to elevated dietary selenium (Se based on the determination of the RNA/DNA ratio in muscle, heat shock protein 70 (Hsp70, and caspase-3/7 in muscle and/or liver tissues. Four semi-purified test diets were prepared by adding different levels of L-selenomethionine (0, 50, 100, and 200 mg/kg diet. The analytical determinations of total Se were 2.2, 19.7, 40.1, and 77.7 mg/kg diet. The sturgeon (initial body weight: 30 ± 2 g; mean ± SEM were raised in indoor tanks provided with flow through freshwater (18–19 °C. There were three replicates for each dietary treatment with 25 fish per replicate. The liver and muscle tissues were collected at 4 and 8 weeks after feeding the test diets. A significant interaction between duration and levels of dietary Se exposures on RNA/DNA ratio in the muscle tissue was detected (P < 0.05. Although there was no significant main effect due to the duration of dietary Se exposures (i.e., 4 weeks versus 8 weeks on muscle RNA/DNA ratio (P ≥ 0.05, the ratio was significantly decreased with increasing dietary Se levels. Significant main effects were caused by the duration and levels of dietary Se exposures on Hsp70 in both the muscle and liver tissues, with significant increases in Hsp70 due to a longer exposure (8 weeks and higher levels (40.1 and 77.7 mg Se/kg diet of dietary Se. The caspase-3/7 activity in the liver were significantly higher in fish fed the diets containing 40.1 and 77.7 mg Se/kg diet than those fed the other diets. The toxic thresholds of Se in the muscle were estimated to be 32.2 and 26.6 mg Se/kg for the depressed specific growth rate and the induced Hsp70 response in muscle, respectively. This result indicated that the Hsp70 response in muscle is a more sensitive biomarker than the SGR of sturgeon for evaluating Se toxicity in white sturgeon. Results of the

  6. 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.

  7. Heat shock proteins on the human sperm surface.

    Science.gov (United States)

    Naaby-Hansen, Soren; Herr, John C

    2010-01-01

    The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

  8. The effect of passive heating on heat shock protein 70 and interleukin-6: a possible treatment tool for metabolic diseases?

    OpenAIRE

    Faulkner, SH; Jackson, S; Fatania, G; Leicht, CA

    2017-01-01

    Exercise and physical activity remain the gold standard methods of enhancing and maintaining health and wellbeing. However, in populations that benefit most from exercise, adherence is often poor and alternatives to exercise are important to bring about health improvements. Recent work suggests a role for passive heating (PH) and heat shock proteins (HSP) in improving cardio-metabolic health. The aim of this study was to investigate the expression of HSP70 and IL-6 in response to either exerc...

  9. 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.

  10. Pifithrin-μ, an inhibitor of heat-shock protein 70, can increase the antitumor effects of hyperthermia against human prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Kazumasa Sekihara

    Full Text Available Hyperthermia (HT improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145 were treated with HT (43°C for 2 h. All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.

  11. 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.

  12. 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.

  13. Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yu-Shan [Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (China); Department of Animal Science, National Ilan University, Ilan, Taiwan (China); Liu, Shih-Jen [Vaccine Research and Development Center, National Health Research Institutes, Miaoli, Taiwan (China); Huang, Su-Chen; Chang, Chao-Chun; Huang, Yi-Chun; Fong, Weng-Lam; Chi, Mau-Shin [Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (China); Chi, Kwan-Hwa, E-mail: m006565@ms.skh.org.tw [Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (China); School of Medicine and Institute of Radiation Science and Image Research, National Yang-Ming Medical University, Taipei, Taiwan (China)

    2012-10-29

    Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C′-PSA or rHsp70C′-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

  14. Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy

    International Nuclear Information System (INIS)

    Wang, Yu-Shan; Liu, Shih-Jen; Huang, Su-Chen; Chang, Chao-Chun; Huang, Yi-Chun; Fong, Weng-Lam; Chi, Mau-Shin; Chi, Kwan-Hwa

    2012-01-01

    Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C′-PSA or rHsp70C′-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

  15. Environmental temperature and stocking density effects on acute phase proteins, heat shock protein 70, circulating corticosterone and performance in broiler chickens

    Science.gov (United States)

    Najafi, Pardis; Zulkifli, Idrus; Amat Jajuli, Nurfarahin; Farjam, Abdoreza Soleimani; Ramiah, Suriya Kumari; Amir, Anna Aryani; O'Reily, Emily; Eckersall, David

    2015-11-01

    An experiment was conducted to determine the effect of different stocking densities on serum corticosterone (CORT), ovotransferrin (OVT), α1-acid glycoprotein (AGP) and ceruloplasmin (CP) concentrations, brain heat shock protein (HSP) 70 expression and performance in broiler chickens exposed to unheated and heated conditions. Day-old chicks were stocked at 0.100 m2/bird (low density (LD)) or 0.063 m2/bird (high density (HD)), in battery cages and housed in environmentally controlled rooms. From 21 to 35 days of age, birds from each stocking density group were exposed to either 24 or 32 °C. Growth performance was recorded during the heat treatment period, and blood and brain samples were collected to determine CORT, OVT, AGP, CP and HSP 70 levels on day 35. Heat treatment but not stocking density was detrimental to growth performance. There were significant temperature × density interactions for CORT, CP and OVT on day 35. Although HD elevated CORT, CP and OVT when compared to LD, the effects of the former were more obvious under heated condition. Both temperature and density had significant effect on AGP and HSP 70. In conclusion, irrespective of temperature, high stocking density was physiologically stressful to broiler chickens, as indicated by CORT, AGP, CP, OVT and HSP 70, but not detrimental to growth performance and survivability. As it was shown in the present study, AGP, CP and OVT could be useful biomarkers to determine the effect of overcrowding and high temperature on the welfare of broiler chickens.

  16. 5,7-Dimethoxycoumarin prevents chronic mild stress induced depression in rats through increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2018-02-01

    Full Text Available The current study was aimed to investigate the role of 5,7-dimethoxycoumarin in the prevention of chronic mild stress induced depression in rats. The chronic mild stress rat model was prepared using the known protocols. The results from open-field test showed that rats in the chronic mild stress group scored very low in terms of crossings and rearings than those of the normal rats. However, pre-treatment of the rats with 10 mg/kg doses of 5,7-dimethoxycoumarin prevented decline in the locomotor activity by chronic mild stress. The level of monoamine oxidase-A in the chronic mild stress rat hippocampus was markedly higher. Chronic mild stress induced increase in the monoamine oxidase-A level was inhibited by pre-treatment with 10 mg/kg doses of 5,7-dimethoxycoumarin in the rats. Chronic mild stress caused a marked increase in the level of caspase-3 mRNA and proteins in rat hippocampus tissues. The increased level of caspase-3 mRNA and protein level was inhibited by treatment of rats with 5,7-dimethoxycoumarin (10 mg/kg. 5,7-Dimethoxycoumarin administration into the rats caused a marked increase in the levels of heat shock protein-70 mRNA and protein. The levels of heat shock protein-70 were markedly lower both in normal and chronic mild stress groups of rats compared to the 5,7-dimethoxycoumarin treated groups. Thus 5,7-dimethoxycoumarin prevented the chronic mild stress induced depression in rats through an increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels.

  17. 5,7-Dimethoxycoumarin prevents chronic mild stress induced depression in rats through increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels.

    Science.gov (United States)

    Yang, Wei; Wang, Huanlin

    2018-02-01

    The current study was aimed to investigate the role of 5,7-dimethoxycoumarin in the prevention of chronic mild stress induced depression in rats. The chronic mild stress rat model was prepared using the known protocols. The results from open-field test showed that rats in the chronic mild stress group scored very low in terms of crossings and rearings than those of the normal rats. However, pre-treatment of the rats with 10 mg/kg doses of 5,7-dimethoxycoumarin prevented decline in the locomotor activity by chronic mild stress. The level of monoamine oxidase-A in the chronic mild stress rat hippocampus was markedly higher. Chronic mild stress induced increase in the monoamine oxidase-A level was inhibited by pre-treatment with 10 mg/kg doses of 5,7-dimethoxycoumarin in the rats. Chronic mild stress caused a marked increase in the level of caspase-3 mRNA and proteins in rat hippocampus tissues. The increased level of caspase-3 mRNA and protein level was inhibited by treatment of rats with 5,7-dimethoxycoumarin (10 mg/kg). 5,7-Dimethoxycoumarin administration into the rats caused a marked increase in the levels of heat shock protein-70 mRNA and protein. The levels of heat shock protein-70 were markedly lower both in normal and chronic mild stress groups of rats compared to the 5,7-dimethoxycoumarin treated groups. Thus 5,7-dimethoxycoumarin prevented the chronic mild stress induced depression in rats through an increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels.

  18. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Johannes [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich (Germany); Hillmer, Andreas S. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany); Högen, Tobias [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); McLean, Pamela J. [Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States); Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)

    2016-08-12

    Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein

  19. 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

  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. Recombinant heat shock protein 70 functional peptide and alpha-fetoprotein epitope peptide vaccine elicits specific anti-tumor immunity.

    Science.gov (United States)

    Wang, Xiao-Ping; Wang, Qiao-Xia; Lin, Huan-Ping; Xu, Bing; Zhao, Qian; Chen, Kun

    2016-11-01

    Alpha-fetoprotein (AFP) is a marker of hepatocellular carcinoma (HCC) and serves as a target for immunotherapy. However, current treatments targeting AFP are not reproducible and do not provide complete protection against cancer. This issue may be solved by developing novel therapeutic vaccines with enhanced immunogenicity that could effectively target AFP-expressing tumors. In this study, we report construction of a therapeutic peptide vaccine by linking heat shock protein 70 (HSP70) functional peptide to the AFP epitope to obtain HSP70-P/AFP-P. This novel peptide was administered into BALB/c mice to observe the effects. Quantification of AFP-specific CD8 + T cells that secrete IFN-γ in these mice via ELISPOT revealed the synergistic effects of HSP70-P/AFP-P with increased numbers of AFP-specific CD8 + T cells. Similarly, ELISA analysis showed increased granzyme B and perforin released by natural killer cells. Moreover, in vitro cytotoxic T-lymphocyte assays and in vivo tumor preventive experiments clearly showed the higher antitumor effects of HSP70-P/AFP-P against AFP-expressing tumors. These results show that treatment of BALB/c mice with HSP70-P/AFP-P induced stronger T-cells responses and improved protective immunity. Our data suggest that HSP70-P/AFP-P may be used as a therapeutic approach in the treatment of AFP-expressing cancers.

  3. 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

  4. Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease.

    Science.gov (United States)

    Trotta, Lucia; Weigt, Kathleen; Schinnerling, Katina; Geelhaar-Karsch, Anika; Oelkers, Gerrit; Biagi, Federico; Corazza, Gino Roberto; Allers, Kristina; Schneider, Thomas; Erben, Ulrike; Moos, Verena

    2017-08-01

    Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei , the proportions of activated effector CD4 + T cells, determined as CD40L + IFN-γ + , were significantly lower in patients with CWD than in healthy controls; CD8 + T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei -specific degranulation, although CD69 + IFN-γ + CD8 + T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei -derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei -derived proteins may contribute to the pathogenesis of CWD. Copyright © 2017 American Society for Microbiology.

  5. Pain Relief with Wet Cupping Therapy in Rats is Mediated by Heat Shock Protein 70 and ß-Endorphin

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    Imam Subadi

    2017-07-01

    Full Text Available Background: Wet cupping therapy is a complementary therapy in pain management. The mechanism of this therapy, however, needs further elucidation. Cells injured by wet cupping therapy seem to stimulate the expression of heat shock protein 70 (HSP70. Its benefit in pain reduction could be mediated by the expression of ß-endorphin. This study aimed at determining the correlation between HSP70 and ß-endorphin after wet cupping therapy. Methods: Sixteen male Wistar rats were divided into control (CG; n=8 and treatment (TG; n=8 groups. The rats in both groups were injected with complete Freund’s adjuvant (CFA at the footpad. In the TG, wet cupping therapy was done at the left and right paralumbar regions 48 hours after the CFA injection. Twenty-four hours after therapy, the hot plate test was done to assess pain threshold. Thereafter, immunohistochemistry from the skin subjected to wet cupping therapy was conducted for HSP70 and ß-endorphin. Results: The expression of HSP70 was significantly higher in the keratinocytes of the TG (20.25±3.53; P<0.001 than in the keratinocytes of the CG (10.50±2.44; P<0.001. The expression of ß-endorphin was significantly higher in the keratinocytes of the TG (22.37±3.52; P<0.001 than in the keratinocytes of the CG (5.12±1.72; P<0.001. The results also revealed a high correlation between HSP70 and ß-endorphin (β=0.864; P<0.001. Pain threshold after wet cupping therapy was significantly higher in the TG (22.81±6.34 s; P=0.003 than in the CG (11.78±3.56 s. Conclusions: The benefit of wet cupping therapy in terms of pain reduction in rats could be mediated by the expression of HSP70 and ß-endorphin.

  6. Pain Relief with Wet Cupping Therapy in Rats is Mediated by Heat Shock Protein 70 and ß-Endorphin.

    Science.gov (United States)

    Subadi, Imam; Nugraha, Boya; Laswati, Hening; Josomuljono, Harjanto

    2017-07-01

    Wet cupping therapy is a complementary therapy in pain management. The mechanism of this therapy, however, needs further elucidation. Cells injured by wet cupping therapy seem to stimulate the expression of heat shock protein 70 (HSP70). Its benefit in pain reduction could be mediated by the expression of ß-endorphin. This study aimed at determining the correlation between HSP70 and ß-endorphin after wet cupping therapy. Sixteen male Wistar rats were divided into control (CG; n=8) and treatment (TG; n=8) groups. The rats in both groups were injected with complete Freund's adjuvant (CFA) at the footpad. In the TG, wet cupping therapy was done at the left and right paralumbar regions 48 hours after the CFA injection. Twenty-four hours after therapy, the hot plate test was done to assess pain threshold. Thereafter, immunohistochemistry from the skin subjected to wet cupping therapy was conducted for HSP70 and ß-endorphin. The expression of HSP70 was significantly higher in the keratinocytes of the TG (20.25±3.53; Pcupping therapy was significantly higher in the TG (22.81±6.34 s; P=0.003) than in the CG (11.78±3.56 s). The benefit of wet cupping therapy in terms of pain reduction in rats could be mediated by the expression of HSP70 and ß-endorphin.

  7. HSF1 and HSF3 cooperatively regulate the heat shock response in lizards.

    Science.gov (United States)

    Takii, Ryosuke; Fujimoto, Mitsuaki; Matsuura, Yuki; Wu, Fangxu; Oshibe, Namiko; Takaki, Eiichi; Katiyar, Arpit; Akashi, Hiroshi; Makino, Takashi; Kawata, Masakado; Nakai, Akira

    2017-01-01

    Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.

  8. Geldanamycin induces production of heat shock protein 70 and partially attenuates ototoxicity caused by gentamicin in the organ of Corti explants

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    Haupt Heidemarie

    2009-09-01

    Full Text Available Abstract Background Heat shock protein 70 (HSP70 protects inner ear cells from damage and death induced by e.g. heat or toxins. Benzoquinone ansamycin antibiotic geldanamycin (GA was demonstrated to induce the expression of HSP70 in various animal cell types. The aim of our study was to investigate whether GA induces HSP70 in the organ of Corti (OC, which contains the auditory sensory cells, and whether GA can protect these cells from toxicity caused by a common aminoglycoside antibiotic gentamicin. Methods To address these questions, we used the OC explants isolated from p3-p5 rats. As a read-out, we used RT-PCR, ELISA and immunofluorescence. Results We found that GA at the concentration of 2 μM efficiently induced HSP70 expression on mRNA and protein level in the OC explants. Confocal microscopy revealed that HSP70 induced by GA is expressed by hair cells and interdental cells of spiral limbus. Preincubation of explants with 2 μM GA prior to adding gentamicin (500 μM significantly reduced the loss of outer but not inner hair cells, suggesting different mechanisms of otoprotection needed for these two cell types. Conclusion GA induced HSP70 in the auditory sensory cells and partially protected them from toxicity of gentamicin. Understanding the molecular mechanisms of GA otoprotection may provide insights for preventative therapy of the hearing loss caused by aminoglycoside antibiotics.

  9. Suppression of heat shock protein 70 by siRNA enhances the antitumor effects of cisplatin in cultured human osteosarcoma cells.

    Science.gov (United States)

    Mori, Yuki; Terauchi, Ryu; Shirai, Toshiharu; Tsuchida, Shinji; Mizoshiri, Naoki; Arai, Yuji; Kishida, Tsunao; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

    2017-09-01

    Although advances in chemotherapy have improved the prognosis for osteosarcoma, some patients do not respond sufficiently to treatment. Heat shock protein 70 (Hsp70) is expressed at high levels in cancer cells and attenuates the therapeutic efficacy of anticancer agents, resulting in a poorer prognosis. This study investigated whether small interfering RNA (siRNA)-mediated inhibition of Hsp70 expression in an osteosarcoma cell line would enhance sensitivity to cisplatin. The expression of Hsp70 with cisplatin treatment was observed by using Western blotting and real-time reverse transcription polymerase chain reaction (RT-PCR). Changes in the IC 50 of cisplatin when Hsp70 was inhibited by siRNA were evaluated. Cisplatin's effectiveness in inducing apoptosis was assessed by assay of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), caspase-3 activity, and mitochondrial membrane potential. Up-regulation of Hsp70 expression was dependent on the concentration of cisplatin. Inhibition of Hsp70 expression significantly reduced the IC 50 of cisplatin. When cisplatin was added to osteosarcoma cells with Hsp70 expression inhibited, a significant increase in apoptosis was demonstrated in TUNEL, caspase-3, and mitochondrial membrane potential assays. Inhibition of Hsp70 expression induced apoptosis in cultured osteosarcoma cells, indicating that Hsp70 inhibition enhanced sensitivity to cisplatin. Inhibition of Hsp70 expression may provide a new adjuvant therapy for osteosarcoma.

  10. 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

  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. Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

    Science.gov (United States)

    Chaikam, Vijay; Karlson, Dale T

    2010-01-01

    The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

  13. 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

  14. 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)

  15. 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)

  16. 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.

  17. 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.

  18. Heat Shock Protein-Inducing Property of Diarylheptanoid Containing Chalcone Moiety from Alpinia katsumadai

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    Joo-Won Nam

    2017-10-01

    Full Text Available A new diarylheptanoid containing a chalcone moiety, katsumain H (1, was isolated from the seeds of Alpinia katsumadai. The structure was elucidated using a combination of 1D/2D NMR spectroscopy and mass spectrometry data analysis. The absolute configurations of C-3, C-5, and C-7 in 1 were assigned based on its optical rotation and after comparing its NMR chemical shifts with those of its diastereoisomers, katsumain E and katsumain F, which were previously isolated from this plant and characterized. In this study, the stimulatory effects of compounds 1 and 2 were evaluated on heat shock factor 1 (HSF1, heat shock protein 27 (HSP27, and HSP70. Compounds 1 and 2 increased the expression of HSF1 (1.056- and 1.200-fold, respectively, HSP27 (1.312- and 1.242-fold, respectively, and HSP70 (1.234- and 1.271-fold, respectively, without increased cytotoxicity.

  19. 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.

  20. Ultraviolet filters and heat shock proteins: effects in Chironomus riparius by benzophenone-3 and 4-methylbenzylidene camphor.

    Science.gov (United States)

    Martín-Folgar, Raquel; Aquilino, Mónica; Ozáez, Irene; Martínez-Guitarte, José-Luis

    2018-01-01

    Benzophenone-3 (BP3) and 4-methylbenzylidene camphor (4MBC) are common ultraviolet filters (UV filters), compounds considered as emergent contaminants, used in different products like plastics and personal care products. The levels of these compounds are rising in the wild, but the effects they have on invertebrates are poorly understood. Chironomus riparius is a benthic insect widely used in toxicology, and several studies have been previously performed in our laboratory to determine the effects these compounds have on this organism at the molecular level. We have shown that UV filters can alter the mRNA levels of heat shock protein 70 (Hsp70), one of the most studied heat shock proteins. Although these proteins are crucial for the survival of organisms, little data is available on the effects these emergent contaminants have on them, especially in invertebrates. Here, we analyzed the transcriptional activity of 12 genes covering the different groups of heat shock protein [Hsp10, Hsp17, Hsp21, Hsp22, Hsp23, Hsp24, Hsp27, Hsp34, Hsp40, Hsp60, Hsc70 (3), and Hsc70 (4)] in response to 0.1 and 1 mg/L concentrations of BP3 and 4MBC at 8 and 24 h. The results showed that some small Hsp (sHsp) genes were altered by these compounds, while the genes of proteins present in mitochondria, Hsp10 and Hsp60, did not change. sHsps are also involved in developmental processes, so the observed variations could be due to the endocrine disruption activity described for these compounds rather than to a stress response.

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

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    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.

  2. 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

  3. Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride.

    Science.gov (United States)

    Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

    2017-09-01

    Acute fluoride (F - ) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F - induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (Hsps-Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F - -intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F - for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F - -intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dose-dependent fashion in F - -treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F - -induced heart failure.

  4. 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.

  5. 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

  6. 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.

  7. Virus-Heat Shock Protein Interaction and a Novel Axis for Innate Antiviral Immunity

    Directory of Open Access Journals (Sweden)

    Michael Oglesbee

    2012-09-01

    Full Text Available Virus infections induce heat shock proteins that in turn enhance virus gene expression, a phenomenon that is particularly well characterized for the major inducible 70 kDa heat shock protein (hsp70. However, hsp70 is also readily induced by fever, a phylogenetically conserved response to microbial infections, and when released from cells, hsp70 can stimulate innate immune responses through toll like receptors 2 and 4 (TLR2 and 4. This review examines how the virus-hsp70 relationship can lead to host protective innate antiviral immunity, and the importance of hsp70 dependent stimulation of virus gene expression in this host response. Beginning with the well-characterized measles virus-hsp70 relationship and the mouse model of neuronal infection in brain, we examine data indicating that the innate immune response is not driven by intracellular sensors of pathogen associated molecular patterns, but rather by extracellular ligands signaling through TLR2 and 4. Specifically, we address the relationship between virus gene expression, extracellular release of hsp70 (as a damage associated molecular pattern, and hsp70-mediated induction of antigen presentation and type 1 interferons in uninfected macrophages as a novel axis of antiviral immunity. New data are discussed that examines the more broad relevance of this protective mechanism using vesicular stomatitis virus, and a review of the literature is presented that supports the probable relevance to both RNA and DNA viruses and for infections both within and outside of the central nervous system.

  8. 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.

  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. Heat shock proteins of higher plants

    International Nuclear Information System (INIS)

    Key, J.L.; Lin, C.Y.; Chen, Y.M.

    1981-01-01

    The pattern of protein synthesis changes rapidly and dramatically when the growth temperture of soybean seedling tissue is increased from 28 0 C (normal) to about 40 0 C (heat shock). The synthesis of normal proteins is greatly decreased and a new set of proteins, heat shock proteins, is induced. The heat shock proteins of soybean consist of 10 new bands on one-dimensional NaDodSO 4 gels; a more complex pattern is observed on two-dimensional gels. when the tissue is returned to 28 0 C after 4 hr at 40 0 C, there is progressive decline in the synthesis of heat shock proteins and reappearance of a normal pattern of synthesis by 3 or 4 hr. In vitro translation of poly(A) + RNAs isolated from tissued grown at 28 and 40 0 C shows that the heat shock proteins are translated from a ndw set of mRNAs induced at 40 0 C; furthermore, the abundant class mRNAs for many of the normal proteins persist even though they are translated weakly (or not at all) in vivo at 40 or 42.5 0 C. The heat shock response in soybean appears similar to the much-studied heat shock phenomenon in Drosophila

  11. Dicty_cDB: Contig-U13887-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available k cognate 70kd protein (44kd atpase n-terminal 270 3e-70 U20256_1( U20256 |pid:none) Ceratitis capitata heat shock-li...io rerio heat shock 70kDa prote... 271 9e-71 NRL( 1NGC ) Heat-shock cognate 70kd protein (44kd atpase n-term...BC041201 |pid:none) Xenopus laevis Heat shock cognate ... 270 2e-70 NRL( 1NGD ) Heat-shock cognate 70kd protein (44kd atpase n-term...hitrite 70kDa heat shoc... 269 4e-70 NRL( 1NGB ) Heat-shock cognate 70kd protein (44kd atpase n-term...( 1NGH ) Heat-shock cognate 70kd protein (44kd atpase n-terminal 268 1e-69 EF655662_1( EF655662 |pid:none) T

  12. Heat shock protein 72: release and biological significance during exercise.

    Science.gov (United States)

    Whitham, Martin; Fortes, Matthew Benjamin

    2008-01-01

    The cumulative stressors of exercise manifest themselves at a cellular level by threatening the protein homeostasis of the cell. In these conditions, Heat Shock Proteins (HSP) are synthesised to chaperone mis-folded and denatured proteins. As such, the intracellular HSP response is thought to aid cell survival in the face of otherwise lethal cellular stress. Recently, the inducible isoform of the 70 Kda heat shock protein family, Hsp72 has been detected in the extracellular environment. Furthermore, the release of this protein into the circulation has been shown to occur in response to a range of exercise bouts. The present review summarises the current research on the exercise Hsp72 response, the possible mediators and mechanisms of extracellular (e)Hsp72 release, and the possible biological significance of this systemic response. In particular, the possible role of eHsp72 in the modulation of immunity during exercise is discussed.

  13. Circulating heat shock proteins in women with a history of recurrent vulvovaginitis.

    Science.gov (United States)

    Giraldo, P C; Ribeiro-Filho, A D; Simões, J A; Neuer, A; Feitosa, S B; Witkin, S S

    1999-01-01

    OBJECTIVE: Predisposing factors influencing recurrences of bacterial vaginosis (BV) or vaginitis from Candida remain unidentified for most women. As a component of studies to determine host susceptibility factors to genital tract infections in women, we measured expression of the 60-kDa and 70-kDa heat shock proteins (hsp60 and hsp70, respectively) in the circulation of women with or without a history of recurrent BV or candidal vaginitis and with or without a current lower genital tract infection. Heat shock protein expression is associated with a down-regulation of pro-inflammatory immune responses that would inhibit microbial infection. METHOD: The investigators measured hsp60 and hsp70, antibodies to these proteins, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory cytokine interleukin-10 (IL-10) in sera by ELISA. The study population consisted of 100 women who attended a gynecology clinic in Campinas, Brazil. Of those, 55 had a history of recurrent vulvovaginitis (RV), while 45 were controls with no such history. Only women who were asymptomatic for at least 1 month were studied. RESULTS: Although all were asymptomatic, clinical and microbiological examination revealed that five of the women with a history of RV and two controls had a current candidal vaginal infection; 16 RV patients and 12 controls had BV; and six RV patients had both BV and candidiasis. Twenty-eight RV patients and 31 controls had no clinical or microbiological detectable vaginal infection. Among the RV patients, hsp60 and hsp70 were more prevalent in those with current BV (40.9% and 50.0%, respectively) or a candidal infection (45.5% and 54.5%) than in women with no current infection (21.4% and 17.9%). In the women with no history of RV, BV was not associated with a high prevalence of hsp60 (8.3%) or hsp70 (8.3%). Interleukin-10 and TNF were not more prevalent in vaginitis patients or controls with a current candidal infection or BV than in

  14. 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.

  15. Guidelines for the nomenclature of the human heat shock proteins

    NARCIS (Netherlands)

    Kampinga, Harm H.; Hageman, Jurre; Vos, Michel J.; Kubota, Hiroshi; Tanguay, Robert M.; Bruford, Elspeth A.; Cheetham, Michael E.; Chen, B.; Hightower, Lawrence E.

    The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40),

  16. Solution structure of GSP13 from Bacillus subtilis exhibits an S1 domain related to cold shock proteins

    International Nuclear Information System (INIS)

    Yu Wenyu; Hu Jicheng; Yu Bingke; Xia Wei; Jin Changwen; Xia Bin

    2009-01-01

    GSP13 encoded by gene yugI is a σ B -dependent general stress protein in Bacillus subtilis, which can be induced by heat shock, salt stress, ethanol stress, glucose starvation, oxidative stress and cold shock. Here we report the solution structure of GSP13 and it is the first structure of S1 domain containing protein in Bacillus subtilis. The structure of GSP13 mainly consists of a typical S1 domain along with a C-terminal 50-residue flexible tail, different from the other known S1 domain containing proteins. Comparison with other S1 domain structures reveals that GSP13 has a conserved RNA binding surface, and it may function similarly to cold shock proteins in response to cold stress

  17. TG2 regulates the heat-shock response by the post-translational modification of HSF1.

    Science.gov (United States)

    Rossin, Federica; Villella, Valeria Rachela; D'Eletto, Manuela; Farrace, Maria Grazia; Esposito, Speranza; Ferrari, Eleonora; Monzani, Romina; Occhigrossi, Luca; Pagliarini, Vittoria; Sette, Claudio; Cozza, Giorgio; Barlev, Nikolai A; Falasca, Laura; Fimia, Gian Maria; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi; Piacentini, Mauro

    2018-05-11

    Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response. © 2018 The Authors.

  18. 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

  19. Validation of Heat Shock Protein 70 as a Tumor-Specific Biomarker for Monitoring the Outcome of Radiation Therapy in Tumor Mouse Models

    Energy Technology Data Exchange (ETDEWEB)

    Bayer, Christine; Liebhardt, Michael E.; Schmid, Thomas E. [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Trajkovic-Arsic, Marija [II Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Hube, Kathrin; Specht, Hanno M. [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Schilling, Daniela [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Clinical Kooperation Group, Innate Immunity in Tumor Biology, HelmholtzZentrum München, Munich (Germany); Gehrmann, Mathias; Stangl, Stefan [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Siveke, Jens T. [II Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Wilkens, Jan J. [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Multhoff, Gabriele, E-mail: Gabriele.multhoff@lrz.tum.de [Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich (Germany); Clinical Kooperation Group, Innate Immunity in Tumor Biology, HelmholtzZentrum München, Munich (Germany)

    2014-03-01

    Purpose: Tumor cells, in contrast to normal cells, frequently overexpress heat shock protein 70 (Hsp70) in the cytosol, present it on their cell surface, and actively release it. Therefore, soluble Hsp70 (sHsp70) was investigated as a potential tumor biomarker for monitoring the outcome of radiation therapy. Methods and Materials: Plasma from mice bearing membrane Hsp70 (mHsp70)-positive FaDu human squamous cell carcinoma of the head and neck and spontaneous pancreatic ductal adenocarcinoma (PDAC) was investigated. A cohort of mice with FaDu tumors (0.32 cm{sup 3}) was irradiated with 30 Gy, and plasma was collected 24 hours after irradiation, after the tumors had shrunk to 50% of their starting volume and after complete remission. sHsp70 levels in the plasma were quantified by enzyme-linked immunosorbent assay. Results: sHsp70 levels were significantly higher in the blood of tumor-bearing mice than that of control animals. A correlation between increasing sHsp70 plasma levels and tumor volume in the range of 0.01 cm{sup 3} to 0.66 cm{sup 3} was observed. Radiation-induced regression of the tumors was associated with significantly decreased sHsp70 levels, which returned to the level of control animals after complete remission. Conclusion: We propose sHsp70 as an innovative biomarker for detecting tumors and for monitoring the clinical outcome of radiation therapy in cancer patients.

  20. 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.

  1. Solution structure of the cold-shock-like protein from Rickettsia rickettsii

    International Nuclear Information System (INIS)

    Gerarden, Kyle P.; Fuchs, Andrew M.; Koch, Jonathan M.; Mueller, Melissa M.; Graupner, David R.; O’Rorke, Justin T.; Frost, Caleb D.; Heinen, Heather A.; Lackner, Emily R.; Schoeller, Scott J.; House, Paul G.; Peterson, Francis C.; Veldkamp, Christopher T.

    2012-01-01

    The solution structure of the cold-shock-like protein from R. rickettsii, the causative agent of Rocky Mountain spotted fever, is reported. Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between β-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded β-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4 kJ mol −1 , has a similar stability when compared with other bacterial cold-shock proteins

  2. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    Science.gov (United States)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  3. 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

  4. Increased circulating heat shock protein 70 (HSPA1A) levels in gestational diabetes mellitus: a pilot study.

    Science.gov (United States)

    Garamvölgyi, Zoltán; Prohászka, Zoltán; Rigó, János; Kecskeméti, András; Molvarec, Attila

    2015-07-01

    Recent data indicate that serum Hsp70 (HSPA1A) levels are increased in type 1 and 2 diabetes mellitus. However, there is no report in the literature on circulating Hsp70 levels in gestational diabetes mellitus. In this pilot study, we measured serum Hsp70 levels in 11 pregnant women with pregestational diabetes, 38 women with gestational diabetes, and 40 healthy pregnant women with ELISA. Plasma glucose levels, serum insulin concentrations, HbA1c values, and the Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) index were also determined. According to our results, serum Hsp70 concentrations were significantly higher in women with pregestational and gestational diabetes mellitus than in healthy pregnant women. In addition, pregestational diabetic women had significantly higher Hsp70 levels than those with gestational diabetes. Furthermore, in the group of women with gestational diabetes mellitus, serum Hsp70 levels showed a significant positive correlation with HbA1c values. However, there was no other relationship between clinical features and metabolic parameters of the study subjects and their serum Hsp70 levels in either study group. In conclusion, we demonstrated for the first time in the literature that serum Hsp70 levels are increased and correlate with HbA1c values in women with gestational diabetes mellitus. Nevertheless, further studies are needed to determine whether circulating Hsp70 plays a causative role in the pathogenesis of gestational diabetes or elevated serum Hsp70 levels are only consequences of the disease.

  5. 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.

  6. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

    International Nuclear Information System (INIS)

    Pierce, Anson; Wei, Rochelle; Halade, Dipti; Yoo, Si-Eun; Ran, Qitao; Richardson, Arlan

    2010-01-01

    Research highlights: → Development of mouse overexpressing native human HSF1 in all tissues including CNS. → HSF1 overexpression enhances heat shock response at whole-animal and cellular level. → HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. → HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1 +/0 ) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1 +/0 mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1 +/0 cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1 +/0 cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  7. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Gabriela Huelgas-Morales

    2016-04-01

    Full Text Available In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

  8. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    Science.gov (United States)

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-04-07

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. Copyright © 2016 Huelgas-Morales et al.

  9. The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

    Science.gov (United States)

    Kagawa, H K; Osipiuk, J; Maltsev, N; Overbeek, R; Quaite-Randall, E; Joachimiak, A; Trent, J D

    1995-11-10

    One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were

  10. 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....

  11. Induction of Heat Shock Protein Expression in Cervical Epithelial Cells by Human Semen

    Directory of Open Access Journals (Sweden)

    J. C. Jeremias

    1999-01-01

    Full Text Available Objective: The 70kD heat shock protein (Hsp70, induced when cells are subjected to environmental stress, prevents the denaturation and incorrect folding of polypeptides and may expedite replication and transmission of DNA and RNA viruses. We analyzed whether messenger RNA (mRNA for Hsp70 was expressed following exposure of a cultured human cervical cell line (HeLa cells to human semen or in cervical cells from sexually active women.

  12. 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

  13. Inhibiting Heat-Shock Protein 90 Reverses Sensory Hypoalgesia in Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Michael J Urban

    2010-07-01

    Full Text Available Increasing the expression of Hsp70 (heat-shock protein 70 can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R, 3R, 4S, 5R-3, 4-dihydroxy-5-methoxy-6, 6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

  14. 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

  15. 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...

  16. 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.

  17. Heat Shock Protein 70 Neutralizes Apoptosis-Inducing Factor

    Directory of Open Access Journals (Sweden)

    Guido Kroemer

    2001-01-01

    Full Text Available Programmed cell death (apoptosis is the physiological process responsible for the demise of superfluous, aged, damaged, mutated, and ectopic cells. Its normal function is essential both for embryonic development and for maintenance of adult tissue homeostasis. Deficient apoptosis participates in cancerogenesis, whereas excessive apoptosis leads to unwarranted cell loss accounting for disparate diseases including neurodegeneration and AIDS. One critical step in the process of apoptosis consists in the permeabilization of mitochondrial membranes, leading to the release of proteins which normally are secluded behind the outer mitochondrial membrane[1]. For example, cytochrome c, which is normally confined to the mitochondrial intermembrane space, is liberated from mitochondria and interacts with a cytosolic protein, Apaf-1, causing its oligomerization and constitution of the so-called apoptosome, a protein complex which activates a specific class of cysteine proteases, the caspases[2]. Another example concerns the so-called apoptosis-inducing factor (AIF, another mitochondrial intermembrane protein which can translocate to the nucleus where it induces chromatin condensation and DNA fragmentation[3].

  18. Heat Shock Proteins 60 and 70 Specific Proinflammatory and Cytotoxic Response of CD4+CD28null Cells in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Ashok K. Yadav

    2013-01-01

    Full Text Available Background. CD4+CD28null T cells are expanded in peripheral blood of patients with chronic kidney disease and associated with subclinical atherosclerosis. However, triggers for the oligoclonal expansion and activation of these cells are not clear. Methods. We investigated twenty-five stage V-IV chronic kidney disease (CKD patients and eight healthy subjects (HC. Peripheral mononuclear cells were isolated and incubated with heat shock protein- (HSP 60 and 70. CD4+CD28null and CD4+CD28+ cells were sorted by flowcytometry and antigen specific response was assessed by the mRNA and protein expression of interferon (IFN-γ, perforin, and granzyme B using qRT-PCR and Elispot. Results. The basal mRNA expression of IFN-γ, perforin, and granzyme B in CD4+CD28null cells was higher in subjects with CKD compared to that in HC (P<0.0001. Subjects with CKD also showed expression of IFN-γ, perforin, and granzyme B in the CD4+CD28+ subset, but this was much weaker than that seen in the CD4+CD28null population (P<0.0001. We did not note the expression of these molecules at mRNA or protein level in either subset of CD4 cells in HC. After incubation with HSP60 and HSP70, CD4+CD28null cells showed increased expression at mRNA (P<0.001 and protein level (P<0.001. CD4+CD28+ cells also showed a weak increase in expression. No antigen-specific response was noted in HC. Conclusion. These data show that CD4+CD28null cells in subjects with CKD react with HSP60 and HSP70 by upregulating the expression of IFN-γ, perforin and granzyme B. Increased circulating level of HSP60 and HSP70 might play a role in initiation and/or progression of atherosclerosis in CKD subjects through perturbation of CD4+CD28null cells.

  19. 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.

  20. Riboflavin protects mice against liposaccharide-induced shock through expression of heat shock protein 25

    Science.gov (United States)

    Riboflavin (vitamin B2) is a water-soluble vitamin essential for normal cellular functions, growth and development. The study was aimed at investigating the effects of vitamin B2 on the survival rate, and expressions of tissue heat shock protein 25 (HSP25) and heat shock factor 1 (HSF1) in mice und...

  1. Cold shock protein YB-1 is involved in hypoxia-dependent gene transcription

    International Nuclear Information System (INIS)

    Rauen, Thomas; Frye, Bjoern C.; Wang, Jialin; Raffetseder, Ute; Alidousty, Christina; En-Nia, Abdelaziz; Floege, Jürgen; Mertens, Peter R.

    2016-01-01

    Hypoxia-dependent gene regulation is largely orchestrated by hypoxia-inducible factors (HIFs), which associate with defined nucleotide sequences of hypoxia-responsive elements (HREs). Comparison of the regulatory HRE within the 3′ enhancer of the human erythropoietin (EPO) gene with known binding motifs for cold shock protein Y-box (YB) protein-1 yielded strong similarities within the Y-box element and 3′ adjacent sequences. DNA binding assays confirmed YB-1 binding to both, single- and double-stranded HRE templates. Under hypoxia, we observed nuclear shuttling of YB-1 and co-immunoprecipitation assays demonstrated that YB-1 and HIF-1α physically interact with each other. Cellular YB-1 depletion using siRNA significantly induced hypoxia-dependent EPO production at both, promoter and mRNA level. Vice versa, overexpressed YB-1 significantly reduced EPO-HRE-dependent gene transcription, whereas this effect was minor under normoxia. HIF-1α overexpression induced hypoxia-dependent gene transcription through the same element and accordingly, co-expression with YB-1 reduced HIF-1α-mediated EPO induction under hypoxic conditions. Taken together, we identified YB-1 as a novel binding factor for HREs that participates in fine-tuning of the hypoxia transcriptome. - Highlights: • Hypoxia drives nuclear translocation of cold shock protein YB-1. • YB-1 physically interacts with hypoxia-inducible factor (HIF)-1α. • YB-1 binds to the hypoxia-responsive element (HRE) within the erythropoietin (EPO) 3′ enhancer. • YB-1 trans-regulates transcription of hypoxia-dependent genes such as EPO and VEGF.

  2. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

    Science.gov (United States)

    Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

    2013-11-19

    JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

  3. Induction of heat-shock proteins and phagocytic function of chicken macrophage following in vitro heat exposure

    International Nuclear Information System (INIS)

    Miller, L.; Qureshi, M.A.

    1992-01-01

    The protein profiles and phagocytic ability of Sephadex-elicited chicken peritoneal macrophages were examined following heat-shock exposure. Macrophage cultures were exposed to various temperatures, time exposures and recovery periods. Densitometric analysis of SDS-PAGE autoradiographs revealed that heat-induced macrophages synthesized three major (23, 70 and 90 kD) heat-shock proteins (HSPs). The optimal temperature and time for induction of these HSPs was 45-46 degrees C for 1 h, with a variable recovery period for each HSP. Macrophages exposed to 45 degrees C for 30 and 60 min were significantly depressed in phagocytosis of uncoated sheep erythrocytes (SE) under 45 degrees C incubation conditions. However, phagocytosis of antibody-coated SE was not affected when compared to 41 degrees C control cultures. Macrophages allowed to recover at 41 degrees C following heat-shock exhibited no alterations in their phagocytic ability for either antibody-coated or uncoated SE. This study suggests that heat shock induces three major HSPs in chicken peritoneal macrophages in addition to maintaining their Fc-mediated phagocytic function while significantly depressing their nonspecific phagocytosis

  4. Brain tumor magnetic targeting and biodistribution of superparamagnetic iron oxide nanoparticles linked with 70-kDa heat shock protein study by nonlinear longitudinal response

    Energy Technology Data Exchange (ETDEWEB)

    Shevtsov, Maxim A., E-mail: shevtsov-max@mail.ru [Institute of Cytology of the Russian Academy of Sciences (RAS), Tikhoretsky Ave. 4, St. Petersburg 194064 (Russian Federation); A.L. Polenov Russian Research Scientific Institute of Neurosurgery, Mayakovsky str. 12, St. Petersburg 191014 (Russian Federation); Nikolaev, Boris P. [Research Institute of Highly Pure Biopreparations, Pudozhskaya str. 12, St. Petersburg 197110 (Russian Federation); Ryzhov, Vyacheslav A. [Petersburg Nuclear Physics Institute, NRC Kurchatov Institute, Gatchina 188300 (Russian Federation); Yakovleva, Ludmila Y. [Research Institute of Highly Pure Biopreparations, Pudozhskaya str. 12, St. Petersburg 197110 (Russian Federation); Dobrodumov, Anatolii V. [Institute of Macromolecular Compounds of the Russian Academy of Sciences (RAS), Bolshoi pr. 31, St. Petersburg 199004 (Russian Federation); Marchenko, Yaroslav Y. [Research Institute of Highly Pure Biopreparations, Pudozhskaya str. 12, St. Petersburg 197110 (Russian Federation); Margulis, Boris A. [Institute of Cytology of the Russian Academy of Sciences (RAS), Tikhoretsky Ave. 4, St. Petersburg 194064 (Russian Federation); Pitkin, Emil [The Wharton School, University of Pennsylvania, 3730 Walnut St., Philadelphia, PA 19104 (United States); Guzhova, Irina V. [Institute of Cytology of the Russian Academy of Sciences (RAS), Tikhoretsky Ave. 4, St. Petersburg 194064 (Russian Federation)

    2015-08-15

    Brain tumor targeting efficiency and biodistribution of the superparamagnetic nanoparticles conjugated with heat shock protein Hsp70 (SPION–Hsp70) were evaluated in experimental glioma model. Synthesized conjugates were characterized using the method of longitudinal nonlinear response of magnetic nanoparticles to a weak ac magnetic field with measurements of second harmonic of magnetization (NLR-M{sub 2}). Cellular interaction of magnetic conjugates was analyzed in 9L glioma cell culture. The biodistribution of the nanoparticles and their accumulation in tumors was assessed by the latter approach as well. The efficacy of Hsp70-conjugates for contrast enhancement in the orthotopic model of 9L glioma was assessed by MR imaging (11 T). Magnetic nanoparticles conjugated with Hsp70 had the relaxivity properties of the MR-negative contrast agents. Morphological observation and cell viability test demonstrated good biocompatibility of Hsp70-conjugates. Analysis of the T{sub 2}-weighted MR scans in tumor-bearing rats demonstrated the high efficacy of Hsp70-conjugates in contrast enhancement of the glioma in comparison to non-conjugated nanoparticles. High contrast enhancement of the glioma was provided by the accumulation of the SPION–Hsp70 particles in the glioma tissue (as shown by the histological assay). Biodistribution analysis by NLR-M{sub 2} measurements evidenced the many-fold increase (~40) in the tumor-to-normal brain uptake ratio in the Hsp70-conjugates treated animals. Biodistribution pattern of Hsp70-decorated nanoparticles differed from that of non-conjugated SPIONs. Coating of the magnetic nanoparticles with Hsp70 protein enhances the tumor-targeting ability of the conjugates that could be applied in the MR imaging of the malignant brain tumors. - Highlights: • Second-harmonic nonlinear magnetic response is used for biodistribution analysis. • NLR-M{sub 2} ensures high sensibility in detection of SPIONs in tissue. • SPION–Hsp70 conjugates

  5. Brain tumor magnetic targeting and biodistribution of superparamagnetic iron oxide nanoparticles linked with 70-kDa heat shock protein study by nonlinear longitudinal response

    International Nuclear Information System (INIS)

    Shevtsov, Maxim A.; Nikolaev, Boris P.; Ryzhov, Vyacheslav A.; Yakovleva, Ludmila Y.; Dobrodumov, Anatolii V.; Marchenko, Yaroslav Y.; Margulis, Boris A.; Pitkin, Emil; Guzhova, Irina V.

    2015-01-01

    Brain tumor targeting efficiency and biodistribution of the superparamagnetic nanoparticles conjugated with heat shock protein Hsp70 (SPION–Hsp70) were evaluated in experimental glioma model. Synthesized conjugates were characterized using the method of longitudinal nonlinear response of magnetic nanoparticles to a weak ac magnetic field with measurements of second harmonic of magnetization (NLR-M 2 ). Cellular interaction of magnetic conjugates was analyzed in 9L glioma cell culture. The biodistribution of the nanoparticles and their accumulation in tumors was assessed by the latter approach as well. The efficacy of Hsp70-conjugates for contrast enhancement in the orthotopic model of 9L glioma was assessed by MR imaging (11 T). Magnetic nanoparticles conjugated with Hsp70 had the relaxivity properties of the MR-negative contrast agents. Morphological observation and cell viability test demonstrated good biocompatibility of Hsp70-conjugates. Analysis of the T 2 -weighted MR scans in tumor-bearing rats demonstrated the high efficacy of Hsp70-conjugates in contrast enhancement of the glioma in comparison to non-conjugated nanoparticles. High contrast enhancement of the glioma was provided by the accumulation of the SPION–Hsp70 particles in the glioma tissue (as shown by the histological assay). Biodistribution analysis by NLR-M 2 measurements evidenced the many-fold increase (~40) in the tumor-to-normal brain uptake ratio in the Hsp70-conjugates treated animals. Biodistribution pattern of Hsp70-decorated nanoparticles differed from that of non-conjugated SPIONs. Coating of the magnetic nanoparticles with Hsp70 protein enhances the tumor-targeting ability of the conjugates that could be applied in the MR imaging of the malignant brain tumors. - Highlights: • Second-harmonic nonlinear magnetic response is used for biodistribution analysis. • NLR-M 2 ensures high sensibility in detection of SPIONs in tissue. • SPION–Hsp70 conjugates effectively target the

  6. Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: Role of 70kDa heat shock protein expression

    Czech Academy of Sciences Publication Activity Database

    Tollarová-Borovanská, Michaela; Lalouette, L.; Košťál, Vladimír

    2009-01-01

    Roč. 30, č. 5 (2009), s. 312-319 ISSN 0143-2044 R&D Projects: GA ČR GA206/07/0269 Institutional research plan: CEZ:AV0Z50070508 Keywords : insect * cold tolerance * heat shock proteins Subject RIV: ED - Physiology Impact factor: 1.074, year: 2009

  7. Analysis of Msx1 and Msx2 transactivation function in the context of the heat shock 70 (Hspa1b) gene promoter.

    Science.gov (United States)

    Zhuang, Fengfeng; Nguyen, Manuel P; Shuler, Charles; Liu, Yi-Hsin

    2009-04-03

    Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role.

  8. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  9. 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.

  10. The Role of the Heat Shock Protein B8 (HSPB8 in Motoneuron Diseases

    Directory of Open Access Journals (Sweden)

    Paola Rusmini

    2017-06-01

    Full Text Available Amyotrophic lateral sclerosis (ALS and spinal and bulbar muscular atrophy (SBMA are two motoneuron diseases (MNDs characterized by aberrant protein behavior in affected cells. In familial ALS (fALS and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC system. The small heat shock protein B8 (HSPB8 is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

  11. Radiosynthesis of [{sup 18}F]fluoromethyldeoxyspergualin for molecular imaging of heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Pradip; Li, King C. [Department of Radiology, Nuclear Medicine Division, Methodist Hospital Research Institute, Weill Cornell Medical College, 6565 Fannin Street, MB1-066, Houston, TX 77030 (United States); Lee, Daniel Y., E-mail: dlee@tmhs.or [Department of Radiology, Nuclear Medicine Division, Methodist Hospital Research Institute, Weill Cornell Medical College, 6565 Fannin Street, MB1-066, Houston, TX 77030 (United States)

    2011-03-15

    To probe the in vivo role of stress response factors in normal physiology and in solid tumors we have designed a stable {sup 18}F-labeled molecular imaging agent based on a ligand for heat shock protein 70 (HSP70). We describe the synthesis of [{sup 18}F] fluorodeoxymethylspergualin ([{sup 18}F]MeDSG) as a new radiopharmaceutical probe using a prosthetic group, [{sup 18}F]SFB, for efficient and rapid radiolabeling. Ongoing molecular imaging studies are under way to detect HSP70 expression in tumors by positron emission tomography.

  12. Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

    Science.gov (United States)

    Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

    2009-01-01

    Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and Heat shock factors may play a contributing role. PMID:19338779

  13. The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender.

    Science.gov (United States)

    Lloyd, R E; Fazeli, A; Watson, P F; Holt, W V

    2012-01-01

    Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 10⁶ and 800 × 10⁶ spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

  14. 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.

  15. Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides.

    Science.gov (United States)

    Majewska, A M; Kordan, W; Koziorowska-Gilun, M; Wysocki, P

    2017-02-01

    Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two-dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78-kDa glucose-regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta-1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis. © 2016 Blackwell Verlag GmbH.

  16. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  17. Role of heat shock proteins in cell apoptosis

    Directory of Open Access Journals (Sweden)

    Arleta Kaźmierczuk

    2010-06-01

    Full Text Available Apoptosis is, apart from necrosis and autophagy, one of the possible cell death mechanisms eliminating needless, not normal or infected cells. This process ensures quantitative and qualitative cell control of organisms. Apoptosis is tightly regulated, it requires both activation of a large number of genes and energy input. Up-to-date two main apoptotic pathways have been recognized – external/receptor and internal, processed with the participation of mitochondria. Heat shock proteins HSPs, the molecules known from their chaperone activity and molecular conservatism, play essential functions in the course of apoptosis. Among that proteins family, i.e. HSP100, 90, 70, 60, 40 and small molecular (sHSP, there are agents mainly protective against programmed cell death. However, in some conditions some of these proteins may promote apoptosis. This review describes different key apoptotic proteins interacting with main members of HSP family and the consequence of these events for cell survival or apoptosis.

  18. Adaptive response in Drosophila melanogaster heat shock proteins mutant strains

    International Nuclear Information System (INIS)

    Shaposhnikov, M.V.; Moskalev, A.A.; Turysheva, E.V.

    2007-01-01

    Complete text of publication follows. The members of the heat shock proteins (Hsp) family function as molecular chaperones and assist intracellular folding of newly synthesized proteins. Also it is possible that molecular chaperones are induced during adaptive response to oxidative stress and radiation. The aim of our research was to exam the role of heat shock proteins in adaptive response to oxidative stress after low dose rate gamma-irradiation in Drosophila melanogaster. Drosophilamelanogaster strains were kindly provided by Bloomington Drosophila Stock Center (University of state of Indiana, Bloomington, USA). We used wild type strain (CS), heat shock protein mutant strains (Hsp22, Hsp70, Hsp83), and heat shock factor mutant strain (Hsf). Strains were chronically exposured to adaptive dose of gamma-irradiation in dose rate of 0.17 cGy/h during all stages of life history (from the embrional stage to the stage of matured imago). The rate of absorbed dose was 60 cGy. For oxidative-stress challenge twodays old flies were starved in empty vials for 6 h and then transferred to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival data were collected after 26 h of treatment. Dead flies were counted daily. The obtained data were subjected to survival analysis by Kaplan and Meier method and presented as survival curves. Statistical analysis was held by non-parametric methods. To test the significance of the difference between the two age distributions Kolmogorov-Smirnov test was applied. Gehan-Braslow- Wilcoxon and Cox-Mantel tests were used for estimation of median life span differences. In addition the minimal and maximal life span, time of 90% death, and mortality rate doubling time (MRDT) were estimated. The obtained results will be discussed in presentation.

  19. 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.

  20. Enzyme-treated asparagus extract promotes expression of heat shock protein and exerts antistress effects.

    Science.gov (United States)

    Ito, Tomohiro; Maeda, Takahiro; Goto, Kazunori; Miura, Takehito; Wakame, Koji; Nishioka, Hiroshi; Sato, Atsuya

    2014-03-01

    A novel enzyme-treated asparagus extract (ETAS) has been developed as a functional material produced from asparagus stem. Studies were conducted to determine the effect of ETAS on heat shock protein 70 (HSP70) expression and alleviation of stress. HeLa cells were treated with ETAS, and HSP70 mRNA and protein levels were measured using a reverse transcription-polymerase chain reaction (RT-PCR) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. ETAS showed significant increases in HSP70 mRNA at more than 0.125 mg/mL and the protein at more than 1.0 mg/mL. The antistress effect was evaluated in a murine sleep-deprivation model. A sleep-deprivation stress load resulted in elevation of blood corticosterone and lipid peroxide concentrations, while supplementation with ETAS at 200 and 1000 mg/kg body weight was associated with significantly reduced levels of both stress markers, which were in the normal range. The HSP70 protein expression level in mice subjected to sleep-deprivation stress and supplemented with ETAS was significantly enhanced in stomach, liver, and kidney, compared to ETAS-untreated mice. A preliminary and small-sized human study was conducted among healthy volunteers consuming up to 150 mg/d of ETAS daily for 7 d. The mRNA expression of HSP70 in peripheral leukocytes was significantly elevated at intakes of 100 or 150 mg/d, compared to their baseline levels. Since HSP70 is known to be a stress-related protein and its induction leads to cytoprotection, the present results suggest that ETAS might exert antistress effects under stressful conditions, resulting from enhancement of HSP70 expression. © 2014 Institute of Food Technologists®

  1. 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.

  2. 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.

  3. Synthesis and thermotolerance of heat shock proteins in Campylobacter jejuni

    International Nuclear Information System (INIS)

    Kim, C.K.; Kim, H.O.; Lee, K.J.

    1991-01-01

    The heat shock responses of Campylobacter jejuni were studied by examination of their survival rates and synthesis of heat shock proteins. When C. jejuni cells were treated at the sublethal temperatures of 48C° for 30 minutes, most of the cells maintained their viabilities and synthesized the heat shock proteins of 90, 73, and 66 kD in molecular weight. By the method of two-dimensional electrophoresis, the heat shock proteins of C. jejuni were identified to be Hsp90, Hsp73, and Hsp66. During the heat shock at 48C°, the heat shock proteins were induced from about 5 minutes after the heat shock treatment. Their synthesis was continued upto 30 minutes, but remarkably retarded after 50 minutes. When C. jejune cells were heat shocked at 51C° for 30 minutes, the survival rates of the cells were decreased by about 10 3 fold and synthesis of heat shock proteins and normal proteins was also generally retarded. The cells exposed to 55C° for 30 minutes died off by more than 10 5 cells and the new protein synthesis was not observed. But when C. jejuni cells were heat-shocked at the sublethal temperature of 48C° for 15 to 20 minutes and then were exposed at the lethal temperature of 55C° for 30 minutes, their viabilities were higher than those exposed at 55C° for 30 minutes without pre-heat shock at 48C°. Therefore, the heat shock proteins synthesized at the sublethal temperature of 48C° in C. jejuni were thought to be responsible for thermotolerance. However, when C. jejuni cells heat-shocked at various ranges of sublethal and lethal temperatures were placed back to the optimum temperature of 42C°, the multiplication patterns of the cells pretreated at different temperatures were not much different each other

  4. 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.

  5. Heat shock proteins and cancer: How can nanomedicine be harnessed?

    Science.gov (United States)

    Sauvage, Félix; Messaoudi, Samir; Fattal, Elias; Barratt, Gillian; Vergnaud-Gauduchon, Juliette

    2017-02-28

    Heat shock protein (hsp90) is an interesting target for cancer therapy because it is involved in the folding and stabilization of numerous proteins, including many that contribute to the development of cancer. It is part of the chaperone machinery that includes other heat shock proteins (hsp70, hsp27, hsp40) and is mainly localized in the cytosol, although many analogues or isoforms can be found in mitochondrion, endoplasmic reticulum and the cell membrane. Many potential inhibitors of hsp90 have been tested for cancer therapy but their usefulness is limited by their poor solubility in water and their ability to reach the target cells and the correct intracellular compartment. Nanomedicine, the incorporation of active molecules into an appropriate delivery system, could provide a solution to these drawbacks. In this review, we explain the rationale for using nanomedicine for this sort of cancer therapy, considering the properties of the chaperone machinery and of the different hsp90 analogues. We present some results that have already been obtained and put forward some strategies for delivery of hsp90 analogues to specific organelles. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. ANTIOXIDANT STATUS AND EXPRESSION OF HEAT SHOCK PROTEIN OF COBALT-TREATED PORCINE OVARIAN GRANULOSA CELLS

    Directory of Open Access Journals (Sweden)

    Marcela Capcarová

    2013-02-01

    Full Text Available The aim of this study was to determine the activity of superoxide dismutase (SOD, total antioxidant status (TAS and expression of heat shock protein 70 (Hsp70 of porcine ovarian granulosa cells cultured in vitro after cobalt (Co administrations. Ovarian granulosa cells were incubated with cobalt sulphate administrations as follows: group E1 (0.09 mg.ml-1, group E2 (0.13 mg.ml-1, group E3 (0.17 mg.ml-1, group E4 (0.33 mg.ml-1, group E5 (0.5 mg.ml-1 and the control group without any additions for 18 h. Co administration developed stress reaction and promoted accumulation of Hsp70 what resulted in increasing activity of SOD. TAS of granulosa cells increased with higher doses of Co whereas low doses had no effect on this parameter. Trace elements can adversely affect animal female reproductive system and its functions, through either direct or indirect effects on oxidative stress induction.

  7. Differential transcript induction of parsley pathogenesis-related proteins and of a small heat shock protein by ozone and heat shock

    International Nuclear Information System (INIS)

    Eckey-Kaltenbach, H.; Kiefer, E.; Grosskopf, E.; Ernst, D.; Sandermann, H. Jr

    1997-01-01

    Parsley (Petroselinum (crispum L.) is known to respond to pathogen attack by the synthesis of furanocoumarins and to UV irradiation by the synthesis of flavone glycosides whereas ozone treatment results in the induction of both pathways. A cDNA library from parsley plants was differentially screened using labelled reverse-transcribed poly(A)+ RNA isolated from ozone-treated parsley plants. This resulted in the isolation of 13 independent cDNA clones representing ozone-induced genes and of 11 cDNA clones representing ozone-repressed genes. DNA sequencing of several clones resulted in the identification of pathogenesis-related protein 1-3 (PR1-3), of a new member of PR1 cDNAs (PRI-4) and of a small heat shock protein (sHSP). Northern blot analyses showed a transient induction of the three mRNA species after ozone fumigation. In contrast, heat shock treatment of parsley plants resulted in an increase of sHSP mRNA whereas no increase for transcripts of PR1-3 and PR1-4 could be observed. This is the first characterized sHSP cDNA clone for plants induced by heat shock, as well as by oxidative stress caused by ozone. (author)

  8. Initiation but no execution - modulation of peripheral blood lymphocyte apoptosis in rheumatoid arthritis - a potential role for heat shock protein 70

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    Chuturgoon Anil A

    2011-11-01

    Full Text Available Abstract Background Rheumatoid arthritis (RA is a chronic autoimmune disease, which causes synovial damage. Persistence of lymphocyte infiltrates in the rheumatoid synovium has been attributed to abnormal apoptosis. While not comprehensively investigated, perturbations in peripheral blood lymphocyte (PBL apoptosis may also be involved in perpetuation of autoimmune processes in RA. Methods We investigated total, CD4+ and CD19+ PBL apoptosis in our study cohort by monitoring the translocation of phosphatidylserine using the Annexin-V assay. To examine the role of death receptor mediated apoptosis as well as activation-induced-cell-death (AICD, PBLs were labeled with CD95/Fas and CD69 markers and enumerated by flow cytometry. Proteolytic activity of initiator and executioner caspases was determined by luminometry. DNA fragmentation assays were used to examine whether apoptotic signals were transduced to the nucleus. Quantitative PCR arrays were used to investigate apoptotic pathways associated with RA-PBLs. Since heat-shock-protein-70 (HSP70 is an inducible protein which modulates apoptotic signals, we determined HSP70 levels by intra-cellular flow cytometry and western blots. Results The RA-PBLs showed signs of elevated apoptosis whilst in circulation. These include increases in the loss of plasma membrane asymmetry, indicated by increased externalization of phosphatidylserine (especially in B-lymphocytes. RA-PBLs showed a bias to CD95/Fas mediated apoptotic pathways, but low levels of the CD69 marker suggested that this was not associated with immune activation. Although downstream markers of apoptosis such as caspase-3/7 activity, were increased, no DNA fragmentation was observed in RA-PBLs. Interestingly, elevated levels of apoptosis did not correlate with absolute lymphocyte counts in RA patients. Levels of HSP70 were highly elevated in RA-PBLs compared to controls. Conclusion The results suggest that while apoptosis may be initiated in RA

  9. Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions

    International Nuclear Information System (INIS)

    Homa, Joanna; Olchawa, Ewa; Stuerzenbaum, Stephen R.; John Morgan, A.; Plytycz, Barbara

    2005-01-01

    This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes. - Metals upregulate stress response proteins in earthworm coelomocytes

  10. Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions

    Energy Technology Data Exchange (ETDEWEB)

    Homa, Joanna [Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL 30-060 Cracow (Poland); Olchawa, Ewa [Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL 30-060 Cracow (Poland); Stuerzenbaum, Stephen R. [Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff Wales CF10 3TL (United Kingdom); John Morgan, A. [Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff Wales CF10 3TL (United Kingdom); Plytycz, Barbara [Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL 30-060 Cracow (Poland)]. E-mail: plyt@zuk.iz.uj.edu.pl

    2005-05-01

    This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes. - Metals upregulate stress response proteins in earthworm coelomocytes.

  11. Toxoplasma gondii 70 kDa heat shock protein: systemic detection is associated with the death of the parasites by the immune response and its increased expression in the brain is associated with parasite replication.

    Directory of Open Access Journals (Sweden)

    Paulo Victor Czarnewski Barenco

    Full Text Available The heat shock protein of Toxoplasma gondii (TgHSP70 is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs systemically, as well as the protein in the brain of resistant (BALB/c and susceptible (C57BL/6 mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.

  12. 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.

  13. Heat Shock Cognate 70 Inhibitor, VER-155008, Reduces Memory Deficits and Axonal Degeneration in a Mouse Model of Alzheimer’s Disease

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    Ximeng Yang

    2018-01-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disorder resulting in structural brain changes and memory impairment. We hypothesized that reconstructing neural networks is essential for memory recovery in AD. Heat shock cognate 70 (HSC70, a member of the heat shock protein family of molecular chaperones, is upregulated in AD patient brains, and recent studies have demonstrated that HSC70 facilitates axonal degeneration and pathological progression in AD. However, the direct effects of HSC70 inhibition on axonal development and memory function have never been investigated. In this study, we examined the effects of a small-molecule HSC70 inhibitor, VER-155008, on axonal morphology and memory function in a mouse model of AD (5XFAD mice. We found that VER-155008 significantly promoted axonal regrowth in amyloid β-treated neurons in vitro and improved object recognition, location, and episodic-like memory in 5XFAD mice. Furthermore, VER-155008 penetrated into the brain after intraperitoneal administration, suggesting that VER-155008 acts in the brain in situ. Immunohistochemistry revealed that VER-155008 reduced bulb-like axonal swelling in the amyloid plaques in the perirhinal cortex and CA1 in 5XFAD mice, indicating that VER-155008 also reverses axonal degeneration in vivo. Moreover, the two main pathological features of AD, amyloid plaques and paired helical filament tau accumulation, were reduced by VER-155008 administration in 5XFAD mice. This is the first report to show that the inhibition of HSC70 function may be critical for axonal regeneration and AD-like symptom reversal. Our study provides evidence that HSC70 can be used as a new therapeutic target for AD treatment.

  14. [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

  15. 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.

  16. Ultrastructural changes and Heat Shock Proteins 70 induced by atmospheric pollution are similar to the effects observed under in vitro heavy metals stress in Conocephalum conicum (Marchantiales--Bryophyta).

    Science.gov (United States)

    Basile, Adriana; Sorbo, Sergio; Conte, Barbara; Cardi, Manuela; Esposito, Sergio

    2013-11-01

    Changes in ultrastructure and induction of Heat Shock Proteins 70 have been studied in Conocephalum conicum (Marchantiales) collected in different urban and country sites in Italy. These results were compared to the effects in vitro of exposition to different heavy metals for several days. At urban sites, cellular ultrastructure was modified, and heavy metals could be observed accumulating in cell walls. Simultaneously, a strong increment in Hsp70 was detected, compared with results observed on control specimens. When C. conicum was exposed to heavy metals in vitro, comparable effects as in polluted sites were observed: Cd and Pb accumulated mostly within parenchyma and, within cells, were absorbed to cell walls or concentrated in vacuoles. Moreover, severe alterations were observed in organelles. Concomitantly, a progressive accumulation of Hsp70 was detected following heavy metals exposition. These effects are discussed in order to describe the dose and time-dependent response to heavy metal stress in C. conicum. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Effects of heat shock protein 90 expression on pectoralis major oxidation in broilers exposed to acute heat stress.

    Science.gov (United States)

    Hao, Y; Gu, X H

    2014-11-01

    This study was conducted to determine the effects of heat shock protein 90 (HSP90) expression on pH, lipid peroxidation, heat shock protein 70 (HSP70), and glucocorticoid receptor (GR) expression of pectoralis major in broilers exposed to acute heat stress. In total, 90 male broilers were randomly allocated to 3 groups: control (CON), heat stress (HS), or geldanamycin treatment (GA). On d 41, the broilers in the GA group were injected intraperitoneally with GA (5 μg/kg of BW), and the broilers in the CON and HS groups were injected intraperitoneally with saline. Twenty-four hours later, the broilers in the CON group were moved to environmental chambers controlled at 22°C for 2 h, and the broilers in the HS and GA groups were moved to environmental chambers controlled at 40°C for 2 h. The pH values of the pectoralis major after 30 min and 24 h of chilling after slaughter of HS and GA broilers were significantly lower (P stress caused significant increases in sera corticosterone and lactic dehydrogenase, the activity of malondialdehyde and superoxide dismutase, the expression of HSP90 and HSP70, and nuclear expression of GR protein in the pectoralis major (P stress induced a significant decrease in GR protein expression in the cytoplasm and GR mRNA expression. Furthermore, the low expression of HSP90 significantly increased levels of lactic dehydrogenase and malondialdehyde and GR protein expression in the cytoplasm under heat stress (P shock protein 90 was positively correlated with corticosterone and superoxide dismutase activities (P < 0.01), and HSP90 mRNA was negatively correlated with pH after chilling for 24 h. The results demonstrated that HSP90 plays a pivotal role in protecting cells from oxidation. ©2014 Poultry Science Association Inc.

  18. Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans.

    Directory of Open Access Journals (Sweden)

    Martin L Duennwald

    Full Text Available How small heat shock proteins (sHsps might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.

  19. Protective effects of heat shock protein 70 on the acute lung injury of rats with heat stroke and its mechanism

    Directory of Open Access Journals (Sweden)

    Yan GENG

    2017-06-01

    Full Text Available Objective To investigate the protective effect of heat shock protein (HSP 70 on the acute lung injury (ALI of rats with heat stroke. Methods Sixty four rats were randomly (by employing a random number table assigned into a sham-heated group (Sham group, heat stress group (HS group, and HS plus gluttamine treatment group (HS+GLN group and HS plus quercet in treatment group (HS+QU group, 16 each. All rats were housed in a artificial climate chamber, with the rats in the sham groups exposed to a temperature of 23℃ and humidity of 55%±5%, while the rats of HS, HS+GLN and HS+QU groups to an ambient temperature of 39℃ and humidity of 65%. During heat stress or sham heating, rectal temperature (Tr, systolic blood pressure (SBP and pulse rate (PR were monitored to observe the difference in heat stress response among the groups. The time point at which the SBP started to drop from the peak level was taken as the point of HS onset. At the onset of HS, heat exposure was terminated, then the rats were immediately removed from the chamber, and returned to room temperature. The rats were scarified 0h and 6h after HS onset respectively. After bronchoalveolar lavage fluid (BALF was collected, the lungs of all animals were harvested for pathological examination of lung injury. The concentrations of IL-1β, TNF-α and IL-6 in BALF and HSP70 in lung homogenate were measured by using an enzyme linked immunosorbent assay kit. Results Compared with HS and HS+QU groups, the rats in HS+GLN group required significantly greater heat load to induce HS (P<0.001, and had longer survival time span after HS onset. Compared with Sham group, the concentration of HSP70 in lung homogenate in HS group increased in a time-dependent manner (P<0.001. In comparison with HS group, the concentration of HSP70 in lung homogenate from HS+GLN group was significantly elevated at each time point (P<0.001, while the treatment with QU significantly inhibited the expression of HSP70 (P<0

  20. 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.

  1. Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jingshan [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Nettleship, Joanne E.; Sainsbury, Sarah [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Saunders, Nigel J. [Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Owens, Raymond J., E-mail: ray@strubi.ox.ac.uk [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The X-ray crystal structure of the cold-shock domain protein from N. meningitidis reveals a strand-exchanged dimer. The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 Å resolution and shown to comprise a dimer formed by the exchange of two β-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K{sub d} of 1.25 µM.

  2. Stress proteins in lymphocytes: Membrane stabilization does not affect the heat shock response

    International Nuclear Information System (INIS)

    Hughes, C.S.; Repasky, E.A.; Subjeck, J.R.

    1987-01-01

    Temperatures which have been used to induce heat shock proteins (hsps) have been at the upper physiologic limit or well above this limit. In addition, little attention has been given to the effects of physiologic heat exposures on hsp induction in lymphocytes. The author examined temperatures between 39 0 C and 41 0 C on protein synthesis in the following lymphoid cell lines and cells: BDK, EL-4, JM, DO.11, and in dispersed lymph nodes and thymic tissues. In these studies, 39.5 0 appears to be the threshold for hsp induction (as distinguished by gel electrophoresis). At this temperature the induction of the major hsps at 70 and 89 kDa are observed. Hsp 89 appears to be the most strongly induced in all cells examined. In JM cells, a human cell line, heat shock also induces hsp 68, the non-constitutive hsp at this size. These temperatures do not depress normal levels of protein synthesis. When stearic acid or cholesterol was added to lymphocyte cultures prior to heating (which stabilize membranes), hsp induction appears to occur in a manner indistinguishable from cells heated in normal media. This suggests that membrane fluidity (as influenced by these agents) does not affect or depress the heat shock response in these cells. Finally, the authors observed that 2-deoxyglucose and other inducers of glucose regulated proteins in fibroblasts also induce the major glucose regulated proteins in lymphocytes

  3. RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

    Directory of Open Access Journals (Sweden)

    Roelien A M Meijering

    Full Text Available The heat shock response (HSR is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1, which binds to conserved heat shock elements (HSE in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP. Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli.Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

  4. Level of heat shock proteins decreases in individuals carrying B-chromosomes in the grasshopper Eyprepocnemis plorans

    DEFF Research Database (Denmark)

    Teruel, M; Sørensen, Jesper Givskov; Loeschcke, Volker

    2010-01-01

    We analyzed the effect of B-chromosome presence on expression level of heat shock protein 70 (Hsp70) in cerebral ganglion and gonad in both males and females of the grasshopper Eyprepocnemis plorans. Two natural Spanish populations, Salobreña (Granada) and Torrox (Málaga) were assayed, the former...... harbouring a neutralized (non-driving) B-chromosome (B2) and the latter a parasitic (driving) B-chromosome (B24). The analysis was performed by Western blotting, immunostaining and densitometric measuring expression level of the Hsp70 family in adult individuals. The results showed that Hsp70 levels...

  5. An overview on the small heat shock proteins

    African Journals Online (AJOL)

    USER

    2010-02-15

    Feb 15, 2010 ... whose expression is increase when cells are exposed to elevated ... shock due to much slower degradation of the protein, .... Plant sHSPs are all encoded by nuclear genes and are .... genesis, germination, pollen growth and fruit maturation). ... Production of high levels of heat shock proteins can also.

  6. 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

  7. Neurotoxicity induced by arsenic in Gallus Gallus: Regulation of oxidative stress and heat shock protein response.

    Science.gov (United States)

    Zhao, Panpan; Guo, Ying; Zhang, Wen; Chai, Hongliang; Xing, Houjuan; Xing, Mingwei

    2017-01-01

    Arsenic, a naturally occurring heavy metal pollutant, is one of the functioning risk factors for neurological toxicity in humans. However, little is known about the effects of arsenic on the nervous system of Gallus Gallus. To investigate whether arsenic induce neurotoxicity and influence the oxidative stress and heat shock proteins (Hsps) response in chickens, seventy-two 1-day-old male Hy-line chickens were treated with different doses of arsenic trioxide (As 2 O 3 ). The histological changes, antioxidant enzyme activity, and the expressions of Hsps were detected. Results showed slightly histology changes were obvious in the brain tissues exposure to arsenic. The activities of Glutathione peroxidase (GSH-Px) and catalase (CAT) were decreased compared to the control, whereas the malondialdehyde (MDA) content was increased gradually along with increase in diet-arsenic. The mRNA levels of Hsps and protein expressions of Hsp60 and Hsp70 were up-regulated. These results suggested that sub-chronic exposure to arsenic induced neurotoxicity in chickens. Arsenic exposure disturbed the balance of oxidants and antioxidants. Increased heat shock response tried to protect chicken brain tissues from tissues damage caused by oxidative stress. The mechanisms of neurotoxicity induced by arsenic include oxidative stress and heat shock protein response in chicken brain tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Inducing Heat Shock Proteins Enhances the Stemness of Frozen-Thawed Adipose Tissue-Derived Stem Cells.

    Science.gov (United States)

    Shaik, Shahensha; Hayes, Daniel; Gimble, Jeffrey; Devireddy, Ram

    2017-04-15

    Extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of adipose tissue-derived stromal/stem cells (ASCs) as well as other cells. Unfortunately, the conclusion one may draw after decades of research utilizing fundamentally similar cryopreservation techniques is that a barrier exists, which precludes full recovery. We hypothesize that agents capable of inducing a subset of heat shock proteins (HSPs) and chaperones will reduce the intrinsic barriers to the post-thaw recovery of ASCs. ASCs were exposed to 43°C for 1 h to upregulate HSPs, and the temporal HSP expression profile postheat shock was determined by performing quantitative polymerase chain reaction (PCR) and western blotting assays. The expression levels of HSP70 and HSP32 were found to be maximum at 3 h after the heat shock, whereas HSP90 and HSP27 remain unchanged. The heat shocked ASCs cryopreserved during maximal HSPs expression exhibited increased post-thaw viability than the nonheat shocked samples. Histochemical staining and quantitative reverse transcription-PCR indicated that the ASC differentiation potential was retained. Thus, suggesting that the upregulation of HSPs before a freezing insult is beneficial to ASCs and a potential alternative to the use of harmful cryoprotective agents.

  9. The functional range of heat shock proteins to combat environmental toxicity

    International Nuclear Information System (INIS)

    Mahmood, K.; Mahmood, Q.; Pervez, A.; Nasreen, S.

    2012-01-01

    Almost all the organisms possess a system to cope with the harsh physiochemical factors of environment. Such a system is based on a group of stress genes, which show rapid responses in form of stress proteins, especially heat shock proteins, when cells are confronted with insult. Heat shock proteins are now known to express in response to variety of toxic and stress conditions including diseases. As a molecular chaperone, against cytotoxicity, these ensure the functional ability of cells by repairing the denatured proteins, cellular structures like cytoskeleton and centrosomes and processes dealing with protein synthesis are stabilized or repaired during a second stress in stress tolerant cells and organisms. In unstressed cells these play an imperative role in the synthesis and transport of normal proteins. Their role in certain diseases reveals their potential application in medical field. Certain Hsp are helpful in coping carcinogenicity caused environmental pollutants and have been suggested to have anti-apoptotic, anti stress and anti-allergic function. Their expression is tissue and species specific with respect to type, intensity and duration of a toxicant. These are developmentally regulated and help in process of differentiation and thus their abnormal regulation impairs the normal development. However, their role as bio marker in risk assessment of environmental pollution warrants further research. Due to broad functional range, therefore, present review is embracing the functional aspects of smaller and Hsp 70 families expressing in animals under toxic conditions. (author)

  10. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    International Nuclear Information System (INIS)

    Hayashi, Kokoro; Kojima, Chojiro

    2010-01-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  11. Heat Shock Proteins as Danger Signals for Cancer Detection

    International Nuclear Information System (INIS)

    Seigneuric, Renaud; Mjahed, Hajare; Gobbo, Jessica; Joly, Anne-Laure; Berthenet, Kevin; Shirley, Sarah; Garrido, Carmen

    2011-01-01

    First discovered in 1962, heat shock proteins (HSPs) are highly studied with about 35,500 publications on the subject to date. HSPs are highly conserved, function as molecular chaperones for a large panel of “client” proteins and have strong cytoprotective properties. Induced by many different stress signals, they promote cell survival in adverse conditions. Therefore, their roles have been investigated in several conditions and pathologies where HSPs accumulate, such as in cancer. Among the diverse mammalian HSPs, some members share several features that may qualify them as cancer biomarkers. This review focuses mainly on three inducible HSPs: HSP27, HPS70, and HSP90. Our survey of recent literature highlights some recurring weaknesses in studies of the HSPs, but also identifies findings that indicate that some HSPs have potential as cancer biomarkers for successful clinical applications.

  12. Extracellular cell stress (heat shock) proteins-immune responses and disease: an overview.

    Science.gov (United States)

    Pockley, A Graham; Henderson, Brian

    2018-01-19

    Extracellular cell stress proteins are highly conserved phylogenetically and have been shown to act as powerful signalling agonists and receptors for selected ligands in several different settings. They also act as immunostimulatory 'danger signals' for the innate and adaptive immune systems. Other studies have shown that cell stress proteins and the induction of immune reactivity to self-cell stress proteins can attenuate disease processes. Some proteins (e.g. Hsp60, Hsp70, gp96) exhibit both inflammatory and anti-inflammatory properties, depending on the context in which they encounter responding immune cells. The burgeoning literature reporting the presence of stress proteins in a range of biological fluids in healthy individuals/non-diseased settings, the association of extracellular stress protein levels with a plethora of clinical and pathological conditions and the selective expression of a membrane form of Hsp70 on cancer cells now supports the concept that extracellular cell stress proteins are involved in maintaining/regulating organismal homeostasis and in disease processes and phenotype. Cell stress proteins, therefore, form a biologically complex extracellular cell stress protein network having diverse biological, homeostatic and immunomodulatory properties, the understanding of which offers exciting opportunities for delivering novel approaches to predict, identify, diagnose, manage and treat disease.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'. © 2017 The Author(s).

  13. 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.

  14. Wheat Mds-1 encodes a heat-shock protein and governs susceptibility towards the Hessian fly gall midge

    Science.gov (United States)

    Plant pests including insects must manipulate plants in order to utilize the nutrition and environment of the host. Here, we show that the heat-shock protein gene Mayetiola destructor susceptibility gene-1 (Mds-1) is a major susceptibility gene in wheat that allows the gall midge M. destructor, com...

  15. Glutamine's protection against cellular injury is dependent on heat shock factor-1.

    Science.gov (United States)

    Morrison, Angela L; Dinges, Martin; Singleton, Kristen D; Odoms, Kelli; Wong, Hector R; Wischmeyer, Paul E

    2006-06-01

    Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1(+/+)) and knockout (HSF-1(-/-)) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1(+/+) cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1(-/-) cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1(-/-) cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation.

  16. 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.

  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. 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.

  19. Temporal patterns of cardiac performance and genes encoding heat shock proteins and metabolic sensors of an intertidal limpet Cellana toreuma during sublethal heat stress.

    Science.gov (United States)

    Zhang, Shu; Han, Guo-dong; Dong, Yun-wei

    2014-04-01

    Intertidal invertebrates develop effective physiological adaptations to cope with the rapidly changing thermal environment in the intertidal zone. In the present study, the temporal patterns of heart rate, protein carbonyl groups, and genes encoding heat shock proteins (hsp70 and hsp90) and metabolic sensors (ampkα, ampkβ and sirt1) were measured to study the effect of sublethal heat stress on the cardiac function, oxidative stress, heat shock response and cellular metabolism of an intertidal limpet Cellana toreuma. All the physiological parameters are sensitive to temperature and duration of heat stress. Spearman correlation analysis revealed that the correlations between heart rate and levels of heat shock proteins mRNA and metabolic sensors mRNA were statistically significant. These results further suggest that cardiac function plays crucial roles in cellular energy metabolism and heat shock responses. The significant increase of protein carbonyl groups at 34°C after 4h exposure indicated that the failure of cardiac function and the increase of anaerobic metabolism partly leads to the increase of protein carbonyl groups. Generally, the physiological responses to heat stress are sensitive to temperature and are energy-consumptive, as indicated by the upregulation of metabolic sensors mRNA. However, the upregulation of heat shock proteins and metabolic sensors at the post-transcriptional level and related functions need to be confirmed in further experiments. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    Science.gov (United States)

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

  1. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    International Nuclear Information System (INIS)

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-01-01

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43 0 C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37 0 C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 10 9 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

  2. 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.

  3. 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.

  4. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

    2010-11-15

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  5. Barcoding heat shock proteins to human diseases : looking beyond the heat shock response

    NARCIS (Netherlands)

    Kakkar, Vaishali; Meister-Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H.

    There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR) - and thus generally restoring the disturbed protein homeostasis associated with such diseases - has often been suggested as a

  6. Nuclear transport of heat shock proteins in stressed cells

    International Nuclear Information System (INIS)

    Chughtai, Zahoor Saeed

    2001-01-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or β-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay I have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei of stationary phase cells. (author)

  7. Nuclear transport of heat shock proteins in stressed cells

    Energy Technology Data Exchange (ETDEWEB)

    Chughtai, Zahoor Saeed

    2001-07-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or {beta}-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-{beta}-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single {beta}-importin gene. With this assay I have identified Nmd5p as a {beta}-importin required to concentrate Star-{beta}-galactosidase in nuclei of stationary phase cells. (author)

  8. 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.

  9. 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.

  10. Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

    Science.gov (United States)

    Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

    2015-01-01

    Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

  11. 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.

  12. 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.

  13. An overview on the small heat shock proteins | Mahmood | African ...

    African Journals Online (AJOL)

    In eukaryotes, different heat shock genes are expressed uncoordinatedly, whereas in prokaryote, heat shock genes form a regulon and appear simultaneously. sHSPs are associated with nuclei, cytoskeleton and membranes. They bind partially to denatured proteins, preventing irreversible protein aggregation during stress.

  14. RNA-Seq-based analysis of cold shock response in Thermoanaerobacter tengcongensis, a bacterium harboring a single cold shock protein encoding gene.

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    Bo Liu

    Full Text Available BACKGROUND: Although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC, can survive from 50° to 80 °C, but has poor natural competence under cold shock at 50 °C. We therefore examined cold shock responses and their effect on natural competence in this bacterium. RESULTS: The transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature. CONCLUSION: Our study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic

  15. Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Rütgers, Mark; Muranaka, Ligia Segatto; Schulz-Raffelt, Miriam; Thoms, Sylvia; Schurig, Juliane; Willmund, Felix; Schroda, Michael

    2017-12-01

    A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered. © 2017 John Wiley & Sons Ltd.

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

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    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.

  17. 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

  18. Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis.

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    Huafei Zou

    Full Text Available Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101 to the caveolin-binding domain (CBD of Ku70 (amino acids 471-478. Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A, limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.

  19. 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.

  20. 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...

  1. 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

  2. Heat Shock Proteins in Tendinopathy: Novel Molecular Regulators

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    Neal L. Millar

    2012-01-01

    Full Text Available Tendon disorders—tendinopathies—are the primary reason for musculoskeletal consultation in primary care and account for up to 30% of rheumatological consultations. Whilst the molecular pathophysiology of tendinopathy remains difficult to interpret the disease process involving repetitive stress, and cellular load provides important mechanistic insight into the area of heat shock proteins which spans many disease processes in the autoimmune community. Heat shock proteins, also called damage-associated molecular patterns (DAMPs, are rapidly released following nonprogrammed cell death, are key effectors of the innate immune system, and critically restore homeostasis by promoting the reconstruction of the effected tissue. Our investigations have highlighted a key role for HSPs in tendion disease which may ultimately affect tissue rescue mechanisms in tendon pathology. This paper aims to provide an overview of the biology of heat shock proteins in soft tissue and how these mediators may be important regulators of inflammatory mediators and matrix regulation in tendinopathy.

  3. Effect of patchouli alcohol on the regulation of heat shock-induced oxidative stress in IEC-6 cells.

    Science.gov (United States)

    Liu, Xiaoxi; Jiang, Linshu; Liu, Fenghua; Chen, Yuping; Xu, Lei; Li, Deyin; Ma, Yunfei; Li, Huanrong; Xu, Jianqin

    2016-08-01

    Purpose Patchouli alcohol (PA) is used to treat gastrointestinal dysfunction. The purpose of this study was to ascertain the function of PA in the regulated process of oxidative stress in rat intestinal epithelial cells (IEC-6). Materials and methods Oxidative stress was stimulated by exposing IEC-6 cells to heat shock (42 °C for 3 h). IEC-6 cells in treatment groups were pretreated with various concentrations of PA (10, 40, and 80 ng/mL) for 3 h before heat shock. Results Heat shock caused damage to the morphology of IEC-6 cells, and increased reactive oxygen species (ROS) level and malondialdehyde (MDA) content. Moreover, mRNA and protein expression by target genes related to oxidative stress in heat shock were also altered. Specifically, the mRNA expression by HSP70, HSP90, GSH-px, NRF2 nd HO-1were all increased, and Nrf2 and Keap1 protein expression were increased after heat shock. However, pretreatment with PA weakened the level of damage to the cellular morphology, and decreased the MDA content caused by heat shock, indicating PA had cytoprotective activities. Pretreatment with PA at high dose significantly increased generation of intracellular ROS. Compared with the heat shock group alone, PA pretreatment significantly decreased the mRNA expression by HSP70, HSP90, SOD, CAT, GSH-px, KEAP1 and HO-1. Furthermore, the high dose of PA significantly increased Nrf2 protein expression, while both the intermediate and high dose of PA significantly increased HO-1 protein expression. Conclusion Heat-shock-induced oxidative stress in IEC-6 cells, and PA could alleviate the Nrf2-Keap1 cellular oxidative stress responses.

  4. 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.

  5. Systemic release of cytokines and heat shock proteins in porcine models of polytrauma and hemorrhage

    Science.gov (United States)

    Baker, Todd A.; Romero, Jacqueline; Bach, Harold H.; Strom, Joel A.; Gamelli, Richard L.; Majetschak, Matthias

    2011-01-01

    Objective To define systemic release kinetics of a panel of cytokines and heat shock proteins (HSP) in porcine polytrauma/hemorrhage models and to evaluate whether they could be useful as early trauma biomarkers. Design and Setting Prospective study in a research laboratory. Subjects Twenty-one Yorkshire pigs. Measurements and Main Results Pigs underwent polytrauma (femur fractures/lung contusion, P), hemorrhage (mean arterial pressure 25-30mmHg, H), polytrauma plus hemorrhage (P/H) or sham procedure (S). Plasma was obtained at baseline, in 5-15min intervals during a 60min shock period without intervention and in 60-120min intervals during fluid resuscitation for up to 300min. Plasma was assayed for IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/IL-23p40, IL-13, IL-17, IL-18, IFNγ, TGFβ, TNFα, HSP40, HSP70 and HSP90 by ELISA. All animals after S, P and H survived (n=5/group). Three of six animals after P/H died. IL-10 increased during shock after P and this increase was attenuated after H. TNFα increased during the shock period after P, H and also after S. P/H abolished the systemic IL-10 and TNFα release and resulted in 20-30% increased levels of IL-6 during shock. As fluid resuscitation was initiated TNFα and IL-10 levels decreased after P, H and P/H, HSP 70 increased after P, IL-6 levels remained elevated after P/H and also increased after P and S. Conclusions Differential regulation of the systemic cytokine release after polytrauma and/or hemorrhage, in combination with the effects of resuscitation, can explain the variability and inconsistent association of systemic cytokine/HSP levels with clinical variables in trauma patients. Insults of major severity (P/H) partially suppress the systemic inflammatory response. The plasma concentrations of the measured cytokines/HSPs do not reflect injury severity or physiological changes in porcine trauma models and are unlikely to be able to serve as useful trauma biomarkers in patients. PMID:21983369

  6. Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

    Science.gov (United States)

    Moghanibashi, Mehdi; Rastgar Jazii, Ferdous; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

    2013-06-01

    Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

  7. Age-Related Decrease in Heat Shock 70-kDa Protein 8 in Cerebrospinal Fluid Is Associated with Increased Oxidative Stress.

    Science.gov (United States)

    Loeffler, David A; Klaver, Andrea C; Coffey, Mary P; Aasly, Jan O; LeWitt, Peter A

    2016-01-01

    Age-associated declines in protein homeostasis mechanisms ("proteostasis") are thought to contribute to age-related neurodegenerative disorders. The increased oxidative stress which occurs with aging can activate a key proteostatic process, chaperone-mediated autophagy. This study investigated age-related alteration in cerebrospinal fluid (CSF) concentrations of heat shock 70-kDa protein 8 (HSPA8), a molecular chaperone involved in proteostatic mechanisms including chaperone-mediated autophagy, and its associations with indicators of oxidative stress (8-hydroxy-2'-deoxyguanosine [8-OHdG] and 8-isoprostane) and total anti-oxidant capacity. We examined correlations between age, HSPA8, 8-OHdG, 8-isoprostane, and total antioxidant capacity (TAC) in CSF samples from 34 healthy subjects ranging from 20 to 75 years of age. Age was negatively associated with HSPA8 (ρ = -0.47; p = 0.005). An age-related increase in oxidative stress was indicated by a positive association between age and 8-OHdG (ρ = 0.61; p = 0.0001). HSPA8 was moderately negatively associated with 8-OHdG (ρ = -0.58; p = 0.0004). Age and HSPA8 were weakly associated with 8-isoprostane and TAC (range of ρ values: -0.15 to 0.16). Our findings in this exploratory study suggest that during healthy aging, CSF HSPA8 may decrease, perhaps due in part to an increase in oxidative stress. Our results also suggest that 8-OHdG may be more sensitive than 8-isoprostane for measuring oxidative stress in CSF. Further studies are indicated to determine if our findings can be replicated with a larger cohort, and if the age-related decrease in HSPA8 in CSF is reflected by a similar change in the brain.

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

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    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.

  9. Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

    Science.gov (United States)

    Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

    2014-06-01

    The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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

  11. 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.

  12. Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.

    Science.gov (United States)

    Kakkar, Vaishali; Meister-Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H

    2014-04-01

    There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review.

  13. Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

    Directory of Open Access Journals (Sweden)

    Vu T A Dang

    Full Text Available Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01, as determined by flow cytometric analysis. Furthermore, we found that TRAIL- natural killer cells that were adoptively transferred into Rag-2-/- γ chain-/- mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05 in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05. In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05. These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

  14. Thermotolerance and Heat-Shock Protein Gene Expression Patterns in Bemisia tabaci (Hemiptera: Aleyrodidae) Mediterranean in Relation to Developmental Stage.

    Science.gov (United States)

    Jiang, Rui; Qi, Lan-Da; Du, Yu-Zhou; Li, Yuan-Xi

    2017-10-01

    Temperature plays an important role in the growth, development, and geographic distribution of insects. There is convincing evidence that heat-shock proteins (HSPs) play important roles in helping organisms adapt to thermal stress. To better understand the physiological and ecological influence of thermal stress on the different development stages of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Mediterranean species (MED), nymphs and adults were shocked with temperatures of 35, 38, and 41℃ for 1 and 2 h, respectively, and the survival rate, fecundity, and developmental duration were investigated in the laboratory. The expression levels of the hsp40, hsp70, and hsp90 genes were assessed using real-time PCR. The results indicate that the survival rates of the nymphs and adults decreased with increased temperature. A 2-h heat shock at 41℃ induced a significant reduction in fecundity in adults and an increase in developmental duration in young nymphs. Hsp90 showed higher temperature responses to thermal stress than hsp40 or hsp70. The expression levels of the hsps in the adults were significantly down-regulated by a 2-h heat shock at 41℃ compared with that by a 1-h treatment. A significant decrease in the expression levels of the hsps also occurred in the adults when the temperature increased from 38 to 41℃ for the 2-h treatment, whereas no significant decrease occurred in the nymphs. Compared with previous studies, we provide some evidence indicating that MED has the potential to adapt to a wider temperature range than the Middle East-Asia Minor 1 species. © The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. 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.

  16. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

    Energy Technology Data Exchange (ETDEWEB)

    Harel-Bellan, A; Brini, A T; Farrar, W L [National Cancer Institute, Frederick, MD (USA); Ferris, D K [Program Resources, Inc., Frederick, MD (USA); Robin, P [Institut Gustave Roussy, Villejuif (France)

    1989-06-12

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

  17. Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

    Science.gov (United States)

    Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

    2012-12-14

    Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

  18. Differential response of heat shock proteins to uphill and downhill exercise in heart, skeletal muscle, lung and kidney tissues.

    Science.gov (United States)

    Lollo, Pablo C B; Moura, Carolina S; Morato, Priscila N; Amaya-Farfan, Jaime

    2013-01-01

    Running on a horizontal plane is known to increase the concentration of the stress biomarker heat-shock protein (HSP), but no comparison of the expression of HSP70 has yet been established between the uphill (predominantly concentric) and downhill (predominantly eccentric) muscle contractions exercise. The objective of the study was to investigate the relationships between eccentric and concentric contractions on the HSP70 response of the lung, kidney, gastrocnemius, soleus and heart. Twenty-four male Wistar weanling rats were divided into four groups: non-exercised and three different grades of treadmill exercise groups: horizontal, uphill (+7%) and downhill (-7% of inclination). At the optimal time-point of six hours after the exercise, serum uric acid, creatine kinase (CK) and lactate dehydrogenase (LDH) were determined by standard methods and HSP70 by the Western blot analysis. HSP70 responds differently to different types of running. For kidney, heart, soleus and gastrocnemius, the HSP70 expression increased, 230, 180, 150 and 120% respectively of the reference (horizontal). When the contraction was concentric (uphill) and compared to downhill the increase in response of HSP70 was greater in 80% for kidney, 75% for gastrocnemius, 60% for soleus and 280% for the heart. Uric acid was about 50% higher (0.64 ± 0.03 mg·dL(-1)) in the uphill group as compared to the horizontal or downhill groups. Similarly, the activities of serum CK and LDH were both 100% greater for both the uphill and downhill groups as compared to the horizontal group (2383 ± 253 and 647.00 ± 73 U/L, respectively). The responsiveness of HSP70 appeared to be quite different depending on the type of tissue, suggesting that the impact of exercise was not restricted to the muscles, but extended to the kidney tissue. The uphill exercise increases HSP70 beyond the eccentric type and the horizontal running was a lower HSP70 responsive stimulus. Key PointsExercise can induce increases in HSP70 in

  19. Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70p

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yunkun [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); McCombs, Debbie; Nagy, Lisa; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

    2006-03-01

    Tom70p is an important translocase of the outer membrane complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tom70p is an important TOM-complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P2{sub 1}, with unit-cell parameters a = 44.89, b = 168.78, c = 83.41 Å, α = 90.00, β = 102.74, γ = 90.00°. There are two Tom70p molecules in one asymmetric unit, which corresponds to a solvent content of approximately 51%. Structure determination by MAD methods is under way.

  20. Hexavalent chromium, a lung carcinogen, confers resistance to thermal stress and interferes with heat shock protein expression in human bronchial epithelial cells.

    Science.gov (United States)

    Abreu, Patrícia L; Cunha-Oliveira, Teresa; Ferreira, Leonardo M R; Urbano, Ana M

    2018-03-16

    Exposure to hexavalent chromium [Cr(VI)], a lung carcinogen, triggers several types of cellular stresses, namely oxidative, genotoxic and proteotoxic stresses. Given the evolutionary character of carcinogenesis, it is tempting to speculate that cells that survive the stresses produced by this carcinogen become more resistant to subsequent stresses, namely those encountered during neoplastic transformation. To test this hypothesis, we determined whether pre-incubation with Cr(VI) increased the resistance of human bronchial epithelial cells (BEAS-2B cells) to the antiproliferative action of acute thermal shock, used here as a model for stress. In line with the proposed hypothesis, it was observed that, at mildly cytotoxic concentrations, Cr(VI) attenuated the antiproliferative effects of both cold and heat shock. Mechanistically, Cr(VI) interfered with the expression of two components of the stress response pathway: heat shock proteins Hsp72 and Hsp90α. Specifically, Cr(VI) significantly depleted the mRNA levels of the former and the protein levels of the latter. Significantly, these two proteins are members of heat shock protein (Hsp) families (Hsp70 and Hsp90, respectively) that have been implicated in carcinogenesis. Thus, our results confirm and extend previous studies showing the capacity of Cr(VI) to interfere with the expression of stress response components.

  1. Heat Tolerance Induction of the Indian Meal Moth (Lepidoptera: Pyralidae) Is Accompanied by Upregulation of Heat Shock Proteins and Polyols.

    Science.gov (United States)

    Kim, Minhyun; Lee, Seunghee; Chun, Yong Shik; Na, Jahyun; Kwon, Hyeok; Kim, Wook; Kim, Yonggyun

    2017-08-01

    The Indian meal moth, Plodia interpunctella, causes massive damage to stored grains and processed foods. Heat treatment has been widely used to control insect pests infesting stored grains. However, heat treatment may result in unsatisfactory control owing to heat tolerance of target insects. This study quantified the heat tolerance and analyzed its induction in P. interpunctella. Susceptibility of P. interpunctella to different high temperatures was assessed in all developmental stages. Heat treatment at 44 °C for 1 h caused significant mortalities to all developmental stages, with late-instar larvae exhibiting the highest tolerance. However, the survivorship to heat treatment was significantly increased by pre-exposure to 37 °C for 30 min. The induction of heat tolerance was accompanied by upregulation of two heat shock proteins of Hsc70 and Hsp90. Trehalose and glycerol concentrations in the hemolymph also increased after pre-exposure to 37 °C for 30 min. RNA interference (RNAi) by specific double-stranded RNAs effectively suppressed the inducible expressions of both Hsc70 and Hsp90 in response to 37 °C for 30 min. Either RNAi of Hsc70 or Hsp90 significantly impaired the heat tolerance induction of P. interpunctella. These results suggest that the induction of heat tolerance in P. interpunctella involves the upregulation of these heat shock proteins and hemolymph polyol levels. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

    Science.gov (United States)

    Yuan, Peijia; D'Lima, Nadia G; Slavoff, Sarah A

    2018-01-09

    Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

  3. Plasminogen and angiostatin interact with heat shock proteins.

    Science.gov (United States)

    Dudani, Anil K; Mehic, Jelica; Martyres, Anthony

    2007-06-01

    Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85-90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin's interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15-20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin's binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.

  4. Isoform composition and stoichiometry of the ∼ 90-kDa heat shock protein associated with glucocorticoid receptors

    International Nuclear Information System (INIS)

    Mendel, D.B.; Orti, E.

    1988-01-01

    The authors observed that the ∼ 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the ∼ 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the ∼ 90-kDa heat shock protein. The observation that TSTA and the ∼ 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested that the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the ∼ 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the ∼ 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free ∼ 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [ 35 S]methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two ∼ 90-kDa non-steroid-binding subunits. The consistency with which a ∼ 1:2 stoichiometric ratio of steroid binding to ∼ 90-kDa protein is observed supports the view that the ∼ 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes

  5. 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, ...

  6. 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.

  7. 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...

  8. 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

  9. 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

  10. Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs

    Energy Technology Data Exchange (ETDEWEB)

    Sundarraj, Kiruthika; Raghunath, Azhwar; Panneerselvam, Lakshmikanthan; Perumal, Ekambaram, E-mail: ekas2009@buc.edu.in

    2017-02-15

    With increased industrial utilization of iron oxide nanoparticles (Fe{sub 2}O{sub 3}-NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe{sub 2}O{sub 3}-NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe{sub 2}O{sub 3}-NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe{sub 2}O{sub 3}-NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe{sub 2}O{sub 3}-NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe{sub 2}O{sub 3}-NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe{sub 2}O{sub 3}-NPs could be an environmental risk factor for reproductive disease. - Highlights: • Fe{sub 2}O{sub 3}-NPs caused adverse effects on the seminal vesicle and prostate gland of mice • Heat shock proteins (Hsp60, 70 and 90) were

  11. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  12. Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner*

    Science.gov (United States)

    Chai, Ryan C.; Kouspou, Michelle M.; Lang, Benjamin J.; Nguyen, Chau H.; van der Kraan, A. Gabrielle J.; Vieusseux, Jessica L.; Lim, Reece C.; Gillespie, Matthew T.; Benjamin, Ivor J.; Quinn, Julian M. W.; Price, John T.

    2014-01-01

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss. PMID:24692538

  13. Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

    Science.gov (United States)

    Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

    2014-05-09

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

  14. 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...

  15. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  16. The correlation between heat-shock protein accumulation and persistence and chilling tolerance in tomato fruit

    International Nuclear Information System (INIS)

    Sabehat, A.; Weiss, D.; Lurie, S.

    1996-01-01

    Heating tomato fruit (Lycopersicon esculentum) for 48 h at 38 degrees C prevented chilling injury from developing after 21 d at 2 degrees C, whereas unheated fruit developed high levels of injury. Although the overall protein pattern as seen by Coomassie blue staining was similar from heated and unheated fruit, some high- and many low-molecular-mass proteins were observed in the heated fruit that were absent or present in reduced amounts in unheated fruit. When fruit were injected with [35S]methionine at harvest and then heated, they accumulated high levels of specific radiolabeled proteins that could still be detected after 21 d at 2 degrees C. If the fruit were held at 20 degrees C after heating, the label in the proteins declined rapidly and these fruit were also sensitive to chilling injury. Hsp70 antibody reacted more strongly with proteins from heated and chilled fruit than with proteins from chilled fruit. Hsp18.1 antibody reacted strongly with proteins from heated fruit but not with those from unheated fruit. A 23-kD protein, highly labeled in heated fruit but not in unheated fruit, had its amino terminus sequenced. To our knowledge, this is the first report showing a relationship between the persistence of heat-shock proteins and chilling tolerance in a plant tissue

  17. Systemic GLIPR1-ΔTM protein as a novel therapeutic approach for prostate cancer.

    Science.gov (United States)

    Karantanos, Theodoros; Tanimoto, Ryuta; Edamura, Kohei; Hirayama, Takahiro; Yang, Guang; Golstov, Alexei A; Wang, Jianxiang; Kurosaka, Shinji; Park, Sanghee; Thompson, Timothy C

    2014-04-15

    GLIPR1 is a p53 target gene known to be downregulated in prostate cancer, and increased endogenous GLIPR1 expression has been associated with increased production of reactive oxygen species, increased apoptosis, decreased c-Myc protein levels and increased cell cycle arrest. Recently, we found that upregulation of GLIPR1 in prostate cancer cells increases mitotic catastrophe through interaction with heat shock cognate protein 70 (Hsc70) and downregulation of Aurora kinase A and TPX2. In this study, we evaluated the mechanisms of recombinant GLIPR1 protein (glioma pathogenesis-related protein 1-transmembrane domain deleted [GLIPR1-ΔTM]) uptake by prostate cancer cells and the efficacy of systemic GLIPR1-ΔTM administration in a prostate cancer xenograft mouse model. GLIPR1-ΔTM was selectively internalized by prostate cancer cells, leading to increased apoptosis through reactive oxygen species production and to decreased c-Myc protein levels. Interestingly, GLIPR1-ΔTM was internalized through clathrin-mediated endocytosis in association with Hsc70. Systemic administration of GLIPR1-ΔTM significantly inhibited VCaP xenograft growth. GLIPR1-ΔTM showed no evidence of toxicity following elimination from mouse models 8 hr after injection. Our results demonstrate that GLIPR1-ΔTM is selectively endocytosed by prostate cancer cells, leading to increased reactive oxygen species production and apoptosis, and that systemic GLIPR1-ΔTM significantly inhibits growth of VCaP xenografts without substantial toxicity. © 2013 UICC.

  18. 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.

  19. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  20. 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.

  1. A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

    Science.gov (United States)

    Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E; Lieberman, Andrew P; Osawa, Yoichi

    2014-11-01

    In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. © 2014 by the Society for Experimental Biology and Medicine.

  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. 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.

  4. 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

  5. Decreased expression of heat shock proteins may lead to compromised wound healing in type 2 diabetes mellitus patients.

    Science.gov (United States)

    Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Mohan, Gyanendra; Chaturvedi, Sunanda; Singh, Kiran

    2015-01-01

    Heat shock proteins (HSPs) are inducible stress proteins expressed in cells exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site of injury and bring about protein homeostasis. Diabetic wounds are hard to heal and inadequate HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU). To analyze the differential expression of HSPs and their downstream molecules in human diabetic wounds compared to control wounds. Expressional levels of HSP27, HSP47 and HSP70 and their downstream molecules like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8 control subjects without diabetes using RT-PCR, western blot and immunohistochemistry. Our study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p value=diabetic wounds. Our study demonstrates that the down regulation of HSPs in diabetic wounds is associated with wound healing impairment in T2DM subjects. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The Role Of Protective Heat Shock Protein 70 And Proinflammatory Heat Shock Protein 60 Toward The Functional Status Of Acute Thrombotic Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Bertha Jean Que

    2015-08-01

    Full Text Available Abstract Clinical experience suggests that the functional status of stroke patients is not directly proportional to the number of risk factors this means that there are other factors that influence the status of functional role. The aim of this study is to explain the changes in levels of HSP70 and HSP60 associated with changes the functional status of stroke which measured with National Institutes of Health Stroke Scale NIHSS in acute thrombotic ischemic stroke. This research is quantitative research is an observational analytic with a longitudinal observational design prospective cohort study and case control. Data was collected by consecutive sampling. Examination of serum levels of HSP 70 HSP60 and assessment of NIHSS done in three times at the same time they are the first day amp8804 48 hours the third day 72 hours and fifth day 120 hours. There is a significant difference P 0.05 levels of HSP 60 and HSP 70 between patients with acute ischemic stroke cases with normal people control. Change dynamic level of HSP70 HSP60 and NIHSS according time of examination there is a significant difference. The first day of HSP 70 levels the third and fifth shaped the decline curve according to the NIHSS improvement while the levels of HSP60 formed a pattern opposite to the NIHSS. Curve levels of HSP70 and HSP60 levels to get to the point value of HSP 60 and HSP70 normal control. In general there was no effect of risk factors on extensive infarction NIHSS HSP70 and HSP60 except the variable age to HSP70 which in the elderly 70-75 years levels of HSP70 is higher than other age groups. Changes in levels of HSP70 and HSP60 follow the pattern of change in NIHSS towards improvement. Therefore HSP70 and HSP60 can serve as a prediction for degree of functional the acute thrombotic ischemic stroke. Risk factors are the cause of stroke but do not affect the NIHSS. Age affects levels of HSP 70. In general HSP60 and HSP70 can be used as a diagnostic and prognostic

  7. 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

  8. Simple, economical heat-shock devices for zebrafish housing racks.

    Science.gov (United States)

    Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

    2011-12-01

    One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

  9. Expression levels of hsc70 and hsp60 are developmentally regulated during B-cell maturation and not associated to childhood c-ALL at presentation or relapse

    DEFF Research Database (Denmark)

    Wehner, Peder Skov; Nielsen, Bendt; Hokland, Marianne

    2003-01-01

    ) or heat shock protein 60 (hsp60) contribute to B-cell differentiation and leukemogenesis. We compared the expression of these hsps in normal peripheral blood (PB) CD19+ B-cells, in pediatric bone marrow (BM) CD19+ CD10+ B-cell precursors (BCPs) from normal donors, and in BCPs from common acute......Heat shock proteins are potent regulators of apoptosis, and so they may also be involved in normal cellular differentiation and cancerogenesis. We used quantitative two-dimensional gel electrophoresis for determining whether either the constitutive chaperonic heat shock cognate protein 70 (hsc70...

  10. Estradiol improves cardiac and hepatic function after trauma-hemorrhage: role of enhanced heat shock protein expression.

    Science.gov (United States)

    Szalay, László; Shimizu, Tomoharu; Suzuki, Takao; Yu, Huang-Ping; Choudhry, Mashkoor A; Schwacha, Martin G; Rue, Loring W; Bland, Kirby I; Chaudry, Irshad H

    2006-03-01

    Although studies indicate that 17beta-estradiol administration after trauma-hemorrhage (T-H) improves cardiac and hepatic functions, the underlying mechanisms remain unclear. Because the induction of heat shock proteins (HSPs) can protect cardiac and hepatic functions, we hypothesized that these proteins contribute to the salutary effects of estradiol after T-H. To test this hypothesis, male Sprague-Dawley rats ( approximately 300 g) underwent laparotomy and hemorrhagic shock (35-40 mmHg for approximately 90 min) followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17beta-estradiol (1 mg/kg body wt) was administered at the end of the resuscitation. Five hours after T-H and resuscitation there was a significant decrease in cardiac output, positive and negative maximal rate of left ventricular pressure. Liver function as determined by bile production and indocyanine green clearance was also compromised after T-H and resuscitation. This was accompanied by an increase in plasma alanine aminotransferase (ALT) levels and liver perfusate lactic dehydrogenase levels. Furthermore, circulating levels of TNF-alpha, IL-6, and IL-10 were also increased. In addition to decreased cardiac and hepatic function, there was an increase in cardiac HSP32 expression and a reduction in HSP60 expression after T-H. In the liver, HSP32 and HSP70 were increased after T-H. There was no change in heart HSP70 and liver HSP60 after T-H and resuscitation. Estradiol administration at the end of T-H and resuscitation increased heart/liver HSPs expression, ameliorated the impairment of heart/liver functions, and significantly prevented the increase in plasma levels of ALT, TNF-alpha, and IL-6. The ability of estradiol to induce HSPs expression in the heart and the liver suggests that HSPs, in part, mediate the salutary effects of 17beta-estradiol on organ functions after T-H.

  11. Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus.

    Science.gov (United States)

    Janardhanan, Jeshina; Joseph Martin, Sherry; Astrup, Elisabeth; Veeramanikandan, R; Aukrust, Pål; Abraham, Ooriapadickal C; Varghese, George M

    2013-11-01

    Scrub typhus is a highly prevalent bacterial infection in India and South Asia that is caused by Orientia tsutsugamushi. The innate immune response to infections is modulated by Toll-like receptors (TLRs) and heat shock proteins (HSPs). This study was done to assess the prevalence and possible association of TLR and HSP polymorphisms in scrub typhus. TLR4 Asp299Gly, TLR4 Thr399Ile, TLR2 Arg753Gln and HSP70-2 A1267G are single-nucleotide polymorphisms (SNPs) that may modulate their activities, and these SNPs were assessed in 137 scrub typhus patients and 134 controls by PCR restriction fragment length polymorphism. We found that the two TLR4 mutations, TLR4 D299G and TLR4T399I, were present in 19.5% and 22% of the study population, respectively, and was in significant linkage disequilibrium with a D' of 0.8. The TLR2 mutation was found to be rare, whereas the HSP A>G mutation was very common (77.5%). Compared with the controls, the prevalence of heterozygous genotype of the TLR4D299G SNP, but not any of the other SNPs, was significantly higher among scrub typhus patients. Further studies using a larger sample size and more candidate genes may better enable in determining the role of these associations in susceptibility and severity of scrub typhus.

  12. Measured Sonic Boom Signatures Above and Below the XB-70 Airplane Flying at Mach 1.5 and 37,000 Feet

    Science.gov (United States)

    Maglieri, Domenic J.; Henderson, Herbert R.; Tinetti, Ana F.

    2011-01-01

    During the 1966-67 Edwards Air Force Base (EAFB) National Sonic Boom Evaluation Program, a series of in-flight flow-field measurements were made above and below the USAF XB-70 using an instrumented NASA F-104 aircraft with a specially designed nose probe. These were accomplished in the three XB-70 flights at about Mach 1.5 at about 37,000 ft. and gross weights of about 350,000 lbs. Six supersonic passes with the F-104 probe aircraft were made through the XB-70 shock flow-field; one above and five below the XB-70. Separation distances ranged from about 3000 ft. above and 7000 ft. to the side of the XB-70 and about 2000 ft. and 5000 ft. below the XB-70. Complex near-field "sawtooth-type" signatures were observed in all cases. At ground level, the XB-70 shock waves had not coalesced into the two-shock classical sonic boom N-wave signature, but contained three shocks. Included in this report is a description of the generating and probe airplanes, the in-flight and ground pressure measuring instrumentation, the flight test procedure and aircraft positioning, surface and upper air weather observations, and the six in-flight pressure signatures from the three flights.

  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. 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

  15. 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.

  16. Initial crystallographic studies of a small heat-shock protein from Xylella fastidiosa

    International Nuclear Information System (INIS)

    Tada, Susely F. S.; Saraiva, Antonio Marcos; Lorite, Gabriela S.; Rosselli-Murai, Luciana K.; Pelloso, Alexandre César; Santos, Marcelo Leite dos; Trivella, Daniela B. B.; Cotta, Mônica A.; Souza, Anete Pereira de; Aparicio, Ricardo

    2012-01-01

    Initial crystallographic studies of the X. fastidiosa small heat-shock protein HSP17.9 are reported. The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9 kDa (HSP17.9). HSP17.9 was found as one of the proteins that are induced during X. fastidiosa proliferation and infection in citrus culture. Recombinant HSP17.9 was crystallized and surface atomic force microscopy experiments were conducted with the aim of better characterizing the HSP17.9 crystals. X-ray diffraction data were collected at 2.7 Å resolution. The crystal belonged to space group P4 3 22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 Å, and is the first small heat-shock protein to crystallize in this space group

  17. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    Science.gov (United States)

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

  18. Hepatitis C Virus E2 Protein Induces Upregulation of IL-8 Pathways and Production of Heat Shock Proteins in Human Thyroid Cells.

    Science.gov (United States)

    Hammerstad, Sara Salehi; Stefan, Mihaela; Blackard, Jason; Owen, Randall P; Lee, Hanna J; Concepcion, Erlinda; Yi, Zhengzi; Zhang, Weijia; Tomer, Yaron

    2017-02-01

    Thyroiditis is one of the most common extrahepatic manifestations of hepatitis C virus (HCV) infection. By binding to surface cell receptor CD81, HCV envelope glycoprotein E2 mediates entry of HCV into cells. Studies have shown that different viral proteins may individually induce host responses to infection. We hypothesized that HCV E2 protein binding to CD81 expressed on thyroid cells activates a cascade of inflammatory responses that can trigger autoimmune thyroiditis in susceptible individuals. Human thyroid cell lines ML-1 and human thyrocytes in primary cell culture were treated with HCV recombinant E2 protein. The expression of major proinflammatory cytokines was measured at the messenger RNA and protein levels. Next-generation transcriptome analysis was used to identify early changes in gene expression in thyroid cells induced by E2. HCV envelope protein E2 induced strong inflammatory responses in human thyrocytes, resulting in production of interleukin (IL)-8, IL-6, and tumor necrosis factor-α. Furthermore, the E2 protein induced production of several heat shock proteins including HSP60, HSP70p12A, and HSP10, in human primary thyrocytes. In thyroid cell line ML-1, RNA sequencing identified upregulation of molecules involved in innate immune pathways with high levels of proinflammatory cytokines and chemokines and increased expression of costimulatory molecules, specifically CD40, known to be a major thyroid autoimmunity gene. Our data support a key role for HCV envelope protein E2 in triggering thyroid autoimmunity through activation of cytokine pathways by bystander mechanisms. Copyright © 2017 by the Endocrine Society

  19. Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration.

    Science.gov (United States)

    Grifoni, Samira C; McKey, Susan E; Drummond, Heather A

    2008-05-01

    Recent studies suggest members of the degenerin (DEG)/epithelial Na(+) channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes.

  20. Deinococcus gobiensis cold shock protein improves salt stress tolerance of escherichia coli

    International Nuclear Information System (INIS)

    Jiang Shijie; Wang Jin; Yang Mingkun; Chen Ming; Zhang Wei; Luo Xuegang

    2013-01-01

    The Deinococcus gobiensis I-0, an extremely radiation-resistant bacterium, isolated from the Gobi, has superior resistance to abiotic stress (e.g radiation, oxidation, dehydration and so on). The two cold-shock proteins encoded by csp1 (Dgo_CA1136) and csp2 (Dgo_PA0041) were identified in the complete genome sequence of D. gobiensis. In this study, we showed that D. gobiensis Csp1 protected Escherichia coli cells against cold shock and other abiotic stresses such as salt and osmotic shocks. The quantitative real-time PCR assay shows that the expression of trehalose synthase (otsA, otsB) was up-regulated remarkably under salt stress in the csp1-expressing strain, while no difference in the expression of the genes involved in trehalose degradation (treB and treC). The results suggested that Csp1 caused the accumulation of the trehalose was a major feature for improving tolerance to salt stress in E. coli. (authors)

  1. 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

  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. 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.

  4. Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans.

    Science.gov (United States)

    Krivoruchko, Anastasia; Storey, Kenneth B

    2010-03-01

    The effects of 20 h of anoxic submergence in cold water and 5 h of aerobic recovery on the heat shock response were analyzed in four organs of the anoxia-tolerant turtle Trachemys scripta elegans. Immunoblotting was used to analyze levels of active and inactive forms of the heat shock transcription factor 1 (HSF1), nuclear translocation of HSF1, and the levels of six heat shock proteins (HSPs). PCR was also used to retrieve the turtle HSF1 nucleotide sequence; its deduced amino acid sequence showed 97% identity with chicken HSF1. White skeletal muscle showed a strong fivefold increase in the amount of active HSF1 under anoxic conditions as well as an 80% increase in nuclear localization. This was accompanied by upregulation of five HSPs by 1.8- to 2.9-fold: Hsp25, Hsp40, Hsp70, Hsc70, and Hsp90, the latter two remained elevated after 5 h of aerobic recovery. Kidney and liver showed little change in active HSF1 content during anoxia and recovery, but a significant increase in the nuclear localization of HSF1 during anoxia. This supported enhanced expression of three HSPs in kidney (Hsp40, Hsc70, and Hsp90) and four in liver (Hsp40, Hsp60, Hsp70, Hsc70). Heart displayed a strong increase in active HSF1 during anoxia and recovery (6.6- to 6.8-fold higher than control) and increased nuclear localization but heart HSP levels did not rise. The data demonstrate organ-specific regulation of HSPs during anoxia exposure and aerobic recovery in T. s. elegans and suggest that the heat shock response is an important aspect of cytoprotection during facultative anaerobiosis, particularly with regard to underwater hibernation of turtles in cold water.

  5. 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.

  6. 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

  7. Lung protein leakage in feline septic shock.

    Science.gov (United States)

    Schützer, K M; Larsson, A; Risberg, B; Falk, A

    1993-06-01

    The aim of the present study was to explore lung microvascular leakage of protein and water in a feline model of septic shock, using a double isotope technique with external gamma camera detection and gravimetric lung water measurements. The experiments were performed on artificially ventilated cats. One group of cats (n = 8) was given an infusion of live Escherichia coli bacteria, and another group (n = 5) served as a control group receiving saline. Plasma transferrin was radiolabeled in vivo with indium-113m-chloride, and erythrocytes were labeled with technetium-99m. The distribution of these isotopes in the lungs was continuously measured with a gamma camera. A normalized slope index (NSI) was calculated, indicative of the transferrin accumulation corrected for changes in local blood volume that reflect protein leakage. In the septic group there was a protein leakage after bacterial infusion, with a NSI of 39 x 10(-4) +/- 5 x 10(-4) min-1 (mean +/- SEM), and the PaO2 diminished from 21 +/- 1 to 9.5 +/- 1 kPa. In control cats a slight protein leakage with a NSI of 9 +/- 10(-4) +/- 2 x 10(-4) min-1 was detected, probably caused by the operative procedure, but PaO2 did not change. Wet-to-dry-weight ratios of postmortem lungs were not significantly different between the groups. It was concluded that an intravenous infusion of live E. coli bacteria induces a lung capillary protein leakage without increased lung water and a concomitantly disturbed gas exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Destabilization and recovery of a yeast prion after mild heat shock.

    Science.gov (United States)

    Newnam, Gary P; Birchmore, Jennifer L; Chernoff, Yury O

    2011-05-06

    Yeast prion [PSI(+)] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI(+)] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI(+)]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI(+)] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI(+)] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI(+)] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. 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...

  10. Induction of thermal shock proteins and changes in radiosensitivity after heat treatment of Bombyx mori L. embryos

    International Nuclear Information System (INIS)

    Agaev, F.A.

    1993-01-01

    The method of gel-electrophoresis was used to study thermal shock protein synthesis in Bombyx mori embryos exposed to a mixture of heat and gamma-radiation. Induction of thermal shock protein synthesis was not inhibited by gamma-radiation. It is suggested that thermal shock proteins are involved embryo radiosensitivity modification

  11. 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.

  12. 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

  13. 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.

  14. The role of heat shock protein 90 in the regulation of tumor cell apoptosis.

    Science.gov (United States)

    Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Belkina, M V; Maroshkina, A N

    2011-02-01

    Programmed death of Jurkat tumor cells was studied under conditions of culturing with 17-AAG selective inhibitor of heat shock protein with a molecular weight of 90 kDa and etoposide. Apoptosis realization was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Activity of caspase-3 was evaluated spectrophotometrically. Inhibition of heat shock protein with a molecular weight of 90 kDa activated the apoptotic program in Jurkat tumor cells and etoposide-induced apoptosis. The heat shock protein with a molecular weight of 90 kDa acted as apoptosis inhibitor in tumor cells.

  15. Blood-flow restricted training leads to myocelullar macrophage infiltration and upregulation of heat-shock proteins, but no apparent muscle damage

    DEFF Research Database (Denmark)

    Nielsen, Jakob L; Aagaard, Per; Prokhorova, Tatyana A

    2017-01-01

    Previous studies indicate that low-load muscle contractions performed under local blood-flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study...... into the intervention (Mid8) and 3 and 10 days after training cessation (Post3,Post10) to examine macrophage (M1/M2) content as well as heat-shock protein (HSP27/70) and tenascin-C expression. Blood samples (1 wk) were collected before and after (0.1-24 h) the first and last training session to examine markers...... of muscle damage (CK), oxidative stress (TAC,GSH) and inflammation (MCP1,IL-6,TNFa). M1-macrophage content increased 108-165% with BFRE and LLE at Post3 (P

  16. Small heat shock protein message in etiolated Pea seedlings under altered gravity

    Science.gov (United States)

    Talalaiev, O.

    Plants are subjected to various environmental changes during their life cycle To protect themselves against unfavorable influences plant cells synthesize several classes of small heat shock proteins sHsp ranging in size from 15 to 30 kDa This proteins are able to enhance the refolding of chemically denatured proteins in an ATP-independent manner in other words they can function as molecular chaperones The potential contribution of effects of space flight at the plant cellular and gene regulation level has not been characterized yet The object of our study is sHsp gene expression in etiolated Pisum sativum seedlings exposed to altered gravity and environmental conditions We designed primers to detect message for two inducible forms of the cytosolic small heat shock proteins sHsp 17 7 and sHsp 18 1 Applying the RT- PCR we explore sHsps mRNA in pea seedling cells subjected to two types of altered gravity achieved by centrifugation from 3 to 8g by clinorotation 2 rpm and temperature elevation 42oC Temperature elevation as the positive control significantly increased PsHspl7 7 PsHspl8 1 expression We investigate the expression of actin it was constant and comparable for unstressed controls for all variants Results are under discussion

  17. Induction of heat shock-like proteins in Vigna sinensis seedlings growing under ultraviolet-B (280-320 nm) enhanced radiation

    International Nuclear Information System (INIS)

    Nedunchezhian, N.; Annamalainathan, K.; Kulandaivelu, G.

    1992-01-01

    The effect of ultraviolet-B (UV-B) enhanced fluorescent radiation on protein profile and protein synthesis has been investigated in Vigna sinensis L. cv. Walp seedlings growing at various temperatures. In seedlings growing at 30°C, UV-B radiation decreased the level of several proteins as seen in Coomassie brilliant blue stained gel. However, fluorography of the same gel indicates induction of three sets of proteins in the range of 70. 53 and 16 k Da. Such induction under UV-B enhanced radiation resembled that found after heat shock treatments. In seedlings at 10 and 20°C, induction of such proteins varied both qualitatively and quantitatively. At 40°C. UV-B enhanced radiation caused a cumulative effect with temperature. Strong induction of specific proteins by UV-B radiation in seedlings growing under normal temperature indicates a possible protective role

  18. Heat Shock Protein 90 Inhibitor Decreases Collagen Synthesis of Keloid Fibroblasts and Attenuates the Extracellular Matrix on the Keloid Spheroid Model.

    Science.gov (United States)

    Lee, Won Jai; Lee, Ju Hee; Ahn, Hyo Min; Song, Seung Yong; Kim, Yong Oock; Lew, Dae Hyun; Yun, Chae-Ok

    2015-09-01

    The 90-kDa heat-shock protein (heat-shock protein 90) is an abundant cytosolic chaperone, and inhibition of heat-shock protein 90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) compromises transforming growth factor (TGF)-β-mediated transcriptional responses by enhancing TGF-β receptor I and II degradation, thus preventing Smad2/3 activation. In this study, the authors evaluated whether heat-shock protein 90 regulates TGF-β signaling in the pathogenesis and treatment of keloids. Keloid fibroblasts were treated with 17-AAG (10 μM), and mRNA levels of collagen types I and III were determined by real-time reverse- transcriptase polymerase chain reaction. Also, secreted TGF-β1 was assessed by enzyme-linked immunosorbent assay. The effect of 17-AAG on protein levels of Smad2/3 complex was determined by Western blot analysis. In addition, in 17-AAG-treated keloid spheroids, the collagen deposition and expression of major extracellular matrix proteins were investigated by means of Masson trichrome staining and immunohistochemistry. The authors found that heat-shock protein 90 is overexpressed in human keloid tissue compared with adjacent normal tissue, and 17-AAG decreased mRNA levels of type I collagen, secreted TGF-ß1, and Smad2/3 complex protein expression in keloid fibroblasts. Masson trichrome staining revealed that collagen deposition was decreased in 17-AAG-treated keloid spheroids, and immunohistochemical analysis showed that expression of collagen types I and III, elastin, and fibronectin was markedly decreased in 17-AAG-treated keloid spheroids. These results suggest that the antifibrotic action of heat-shock protein 90 inhibitors such as 17-AAG may have therapeutic effects on keloids.

  19. The heat shock proteins at the increasing of the radioresistance of silkworm embryo Bombyx Moril

    International Nuclear Information System (INIS)

    Agaev, F.A.; Garibov, A.A.; Aliev, D.I.; Alieva, I.N.

    2002-01-01

    The aim of this study is revealing the role of Heat Shock Proteins (HSP) at the radio-modification effect of Heat Shock (HS) on the silkworm embryo. Our preliminary study was indicated that 3-daily silkworm embryo is more than 10-12 times sensitive to gamma-radiation in comparative to 7-daily embryo. Investigation of the HS effect on the radiosensitivity of the embryo indicates that thermal treatment (40 deg. C) of 3 daily embryo during 60 min before irradiation leads the increasing of its radioresistance. At the same time, the HS treatment immediately after irradiation capable increases strike affect of irradiation. LD 50 for heat treated embryo before and after irradiation consists of 46.7 and 19.0 Gy, correspondingly. For embryo irradiated without heat treatment LD 50 is 29.5 Gy. Identical effects are observed for 7-daily embryo. Increasing of the radioresistance by HS before irradiation, obviously, may be explained by the embryo cell reply to the stress factor and capable initiated by different biochemical shifts, for example, by induction of HPS synthesis. According to the results of carried out experiments HS treatment leads to the induction or increasing of the HSP synthesis into this embryo. Protein with molecular mass 70 kDa (HSP-70 kDa) has been synthesised de novo. The synthesis of the other two proteins (HSP-83 kDa and HSP-68 kDa) significantly increases at the high temperature. It is noted, that HSP-70 kDa consists of 55-60 % of whole included radioactive mark. Identical induction was observed in the experiments at the combined effect both HS and gamma-radiation on the embryo. At the post-radiation heat treatment the induction of HSP synthesis is observed, too. It was concluded that damages induced by irradiation can not prevent HSP induction into embryo. The result of comparative analysis was shown that in 3-daily embryo in spite of 7-daily embryo the synthesis of HSP is more intensive and correlated with the radio- modification effect of HS. The

  20. Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes.

    Science.gov (United States)

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

    1996-12-10

    Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of alpha-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.

  1. Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in Escherichia coli O157:H7▿

    Science.gov (United States)

    Burt, Sara A.; van der Zee, Ruurd; Koets, Ad P.; de Graaff, Anko M.; van Knapen, Frans; Gaastra, Wim; Haagsman, Henk P.; Veldhuizen, Edwin J. A.

    2007-01-01

    The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7. PMID:17526792

  2. Chaperone activity of human small heat shock protein-GST fusion proteins.

    Science.gov (United States)

    Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

    2017-07-01

    Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

  3. Acid-regulated proteins induced by Streptococcus mutans and other oral bacteria during acid shock.

    Science.gov (United States)

    Hamilton, I R; Svensäter, G

    1998-10-01

    Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the

  4. Heat Shock Protein 90 Inhibitor (17-AAG) Induces Apoptosis and Decreases Cell Migration/Motility of Keloid Fibroblasts.

    Science.gov (United States)

    Yun, In Sik; Lee, Mi Hee; Rah, Dong Kyun; Lew, Dae Hyun; Park, Jong-Chul; Lee, Won Jai

    2015-07-01

    The regulation of apoptosis, proliferation, and migration of fibroblasts is altered in keloids. The 90-kDa heat shock protein (heat shock protein 90) is known to play a key role in such regulation. Therefore, the authors investigated whether the inhibition of heat shock protein 90 in keloid fibroblasts could induce apoptosis and attenuate keloid fibroblast proliferation and migration. The authors evaluated heat shock protein 90 expression in keloid tissues with immunohistochemistry. The authors used cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and annexin V/propidium iodide staining for apoptosis, a wound healing model and cell tracking system to assess cell migration, and Akt Western blotting analysis in keloid fibroblasts after inhibition of heat shock protein 90 with 17-allylaminodemethoxygeldanamycin (17-AAG). The expression of heat shock protein 90 in keloid tissues was significantly increased compared with normal tissues. The 17-AAG-treated keloid fibroblasts showed significantly decreased proliferation, promotion of apoptosis, and decreased expression of Akt. Furthermore, a dose-dependent decrease in cell migration was noted after 17-AAG treatment of keloid fibroblasts. The 17-AAG-treated keloid fibroblasts had less directionality to the wound center and migrated a shorter distance. The authors confirmed that the inhibition of heat shock protein 90 in keloid fibroblasts could promote apoptosis and attenuate proliferation and migration of keloid fibroblasts. Therefore, the authors think that the inhibition of heat shock protein 90 is a key factor in the regulation of biological processes in keloids. With further preclinical study, the authors will be able to apply these results clinically for keloid treatment.

  5. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.

    Science.gov (United States)

    Svensson, Katrin J; Christianson, Helena C; Wittrup, Anders; Bourseau-Guilmain, Erika; Lindqvist, Eva; Svensson, Lena M; Mörgelin, Matthias; Belting, Mattias

    2013-06-14

    The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.

  6. 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

    Science.gov (United States)

    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

  7. The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Chen, Han-Jou; Mitchell, Jacqueline C; Novoselov, Sergey; Miller, Jack; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline A; Cheetham, Michael E; Shaw, Christopher E

    2016-05-01

    Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2, and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with

  8. The dynamics of heat shock system activation in Monomac-6 cells upon Helicobacter pylori infection.

    Science.gov (United States)

    Pierzchalski, P; Jastrzebska, M; Link-Lenczowski, P; Leja-Szpak, A; Bonior, J; Jaworek, J; Okon, K; Wojcik, P

    2014-12-01

    Immune system cells, particularly phagocytes, are exposed to direct contact with pathogens. Because of its nature - elimination of pathogenes - their cytoprotective systems supposed to be quick and forceful. Physiological consequence of phagocytosis for the phagocyte is the apoptotic death to prevent the eventual survival of bacteria as intracellular parasites. However, in some cases, defense systems used by the bacteria force the immune cells to prolong the contact with the pathogen for its effective elimination. Experiments were performed on Monomac-6 cells exposed to live CagA, VacA expressing Helicobacter pylori (H. pylori) over different period of time. Total cellular RNA, cytoplasmic and nuclear proteins were isolated for polymerase chain reaction, Western-blot and electrophoretic mobility shift assay, respectively. We found that Monomac-6 cells infection with H. pylori resulted in the translocation of the entire cellular content of the heat shock protein 70 (HSP70) into the cytoplasm, where its presence could protect cell against toxic products of engulfed bacteria and premature apoptosis. At the same time the nuclear translocation of heat shock factor 1 (HSF-1) and activation of HSP70 gene transcription was noticed. Action of HSP70 might to postpone monocyte apoptosis through protecting cytoplasmic and nuclear proteins from damaging effect of bacterial products, what could be the defending mechanism against the toxic stress caused by engulfed bacteria and provide the immune cell with the sufficient amount of time required for neutralization of the bacteria from phagosomes, even at the expense of temporary lack of the protection of nuclear proteins.

  9. Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

    Science.gov (United States)

    Germot, Agnès; Philippe, Hervé; Le Guyader, Hervé

    1996-01-01

    Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria. PMID:8962101

  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. Nitric oxide-heat shock protein axis in menopausal hot flushes: neglected metabolic issues of chronic inflammatory diseases associated with deranged heat shock response.

    Science.gov (United States)

    Miragem, Antônio Azambuja; Homem de Bittencourt, Paulo Ivo

    2017-09-01

    Although some unequivocal underlying mechanisms of menopausal hot flushes have been demonstrated in animal models, the paucity of similar approaches in humans impedes further mechanistic outcomes. Human studies might show some as yet unexpected physiological mechanisms of metabolic adaptation that permeate the phase of decreased oestrogen levels in both symptomatic and asymptomatic women. This is particularly relevant because both the severity and time span of hot flushes are associated with increased risk of chronic inflammatory disease. On the other hand, oestrogen induces the expression of heat shock proteins of the 70 kDa family (HSP70), which are anti-inflammatory and cytoprotective protein chaperones, whose expression is modulated by different types of physiologically stressful situations, including heat stress and exercise. Therefore, lower HSP70 expression secondary to oestrogen deficiency increases cardiovascular risk and predisposes the patient to senescence-associated secretory phenotype (SASP) that culminates in chronic inflammatory diseases, such as obesities, type 2 diabetes, neuromuscular and neurodegenerative diseases. This review focuses on HSP70 and its accompanying heat shock response (HSR), which is an anti-inflammatory and antisenescent pathway whose intracellular triggering is also oestrogen-dependent via nitric oxide (NO) production. The main goal of the manuscript was to show that the vasomotor symptoms that accompany hot flushes may be a disguised clue for important neuroendocrine alterations linking oestrogen deficiency to the anti-inflammatory HSR. Results from our own group and recent evidence on hypothalamic control of central temperature guided a search on PubMed and Google Scholar websites. Oestrogen elicits rapid production of the vasodilatory gas NO, a powerful activator of HSP70 expression. Whence, part of the protective effects of oestrogen over cardiovascular and neuroendocrine systems is tied to its capacity of inducing the NO

  12. Early Response of Protein Quality Control in Gills Is Associated with Survival of Hypertonic Shock in Mozambique tilapia

    Science.gov (United States)

    Tang, Cheng-Hao; Lee, Tsung-Han

    2013-01-01

    The protein quality control (PQC) mechanism is essential for cell function and viability. PQC with proper biological function depends on molecular chaperones and proteases. The hypertonicity-induced protein damage and responses of PQC mechanism in aquatic organisms, however, are poorly understood. In this study, we examine the short-term effects of different hypertonic shocks on the levels of heat shock proteins (HSPs, e.g., HSP70 and HSP90), ubiquitin-conjugated proteins and protein aggregation in gills of the Mozambique tilapia (Oreochromis mossambicus). Following transfer from fresh water (FW) to 20‰ hypertonicity, all examined individuals survived to the end of experiment. Moreover, the levels of branchial HSPs and ubiquitin-conjugated proteins significantly increased at 3 and 24 h post-transfer, respectively. Up-regulation of HSPs and ubiquitin-conjugated proteins was sufficient to prevent the accumulation of aggregated proteins. However, the survival rate of tilapia dramatically declined at 5 h and all fish died within 7 h after direct transfer to 30‰ hypertonicity. We presumed that this result was due to the failed activation of gill PQC system, which resulted in elevating the levels of aggregated proteins at 3 and 4 h. Furthermore, in aggregated protein fractions, the amounts of gill Na+/K+-ATPase (NKA) remained relatively low when fish were transferred to 20‰ hypertonicity, whereas abundant NKA was found at 4 h post-transfer to 30‰ hypertonicity. This study demonstrated that the response of PQC in gills is earlier than observable changes in localization of ion-secreting transport proteins upon hypertonic challenge. To our knowledge, this is the first study to investigate the regulation of PQC mechanism in fish and characterize its important role in euryhaline teleost survival in response to hypertonic stress. PMID:23690986

  13. 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.

  14. Influence of Xylella fastidiosa cold shock proteins on pathogenesis in grapevine.

    Science.gov (United States)

    Cold shock proteins (CSPs), a family of nucleic acid binding proteins are an essential part of microbial adaptation to temperature changes. Bacterial CSPs are often expressed in a temperature-dependent manner, and act as chaperones, facilitating translation at low temperature by stabilizing mRNA. In...

  15. 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.

  16. DYNAMICS OF HEAT SHOCK PROTEIN-70 SYNTHESIS IN LUNGS DEPENDS ON THE STAGE OF EXPERIMENTAL RESPIRATORY DISTRESS SYNDROME

    Directory of Open Access Journals (Sweden)

    E. V. Prutkina

    2013-01-01

    Full Text Available Abstract. Acute respiratory distress syndrome (ARDS was reproduced in a rat model, by means of intratracheal instillation of granulocyte lysates (a method protected by Russian patent. Expression of HSP-70 in lung cells was determined by immunohistochemical technique at each ARDS stage. A significant increase of HSP-70 expression by all cell types was revealed during exudative stage, being more intensive in alveolocytes type 1, and less expressed in endothelium. During proliferative stage of the disorder, a decreased HSP-70 expression was noted in all cell populations. At these terms, it proved to be high in neutrophils and alveveolocytes type 1, whereas lower expression was registered in endothelium. At fibrotic stage, HSP-70 synthesis remained at high levels in neutrophils, macrophages, fibroblasts and alveolocytes type 1. Endothelium and alveolocytes type 2 exhibited a recurrent increase at fibrotic stage of ARDS, however it did not reach the values typical to the initial stage of the syndrome.

  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. Comparison of procalcitonin and high-sensitivity C-reactive protein for the diagnosis of sepsis and septic shock in the oldest old patients.

    Science.gov (United States)

    Zhang, Hongmin; Wang, Xiaoting; Zhang, Qing; Xia, Ying; Liu, Dawei

    2017-08-01

    Although the role of serum procalcitonin (PCT) and high-sensitivity C-reactive protein (hs-CRP) in the diagnosis of sepsis and septic shock is well studied, it has not been investigated among oldest old patients. The aim of our study is to determine the role of PCT and hs-CRP in the assessment of sepsis and septic shock in this specific group of patients in the ICU. This is a prospective observational study. Patients >85 years of age admitted to the ICU from May 1st, 2016 to February 1st, 2017 were evaluated. Patients were divided into a sepsis and septic shock group(sepsis/SS) and a non-sepsis group. Serum levels of PCT, hs-CRP and the WBC were measured within 12 h of admission. A total of 70 patients aged 85 years and older were enrolled in this study. Fifty patients were labelled as sepsis/SS and the other 20 were labelled non-sepsis. A ROC analysis showed that the area under the curves (AUC) of hs-CRP and PCT for the discrimination of sepsis/SS patients were 0.825 (95% confidence interval[CI]: 0.73-0.92; P sepsis/SS group, 27 patients had sepsis, while the other 23 patients had septic shock. The ROC analysis showed that the AUCs of hs-CRP and PCT for the discrimination of septic shock patients from sepsis patients were 0.751 (95% CI: 0.62-0.88; P = 0.002) and 0.719 (95% CI:0.57-0.86; p = 0.007), respectively. For the oldest old patients, hs-CRP is not inferior to PCT in the diagnosis of sepsis and septic shock.

  19. 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

  20. 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.

  1. The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

    Directory of Open Access Journals (Sweden)

    Vladislav Kljashtorny

    Full Text Available The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD. Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP, albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

  2. Particle acceleration at quasi-perpendicular shock waves: Theory and observations at 1 AU

    International Nuclear Information System (INIS)

    Parker, L. Neergaard; Zank, G. P.; Hu, Q.

    2014-01-01

    The injection of particles into the diffusive shock acceleration mechanism at highly perpendicular (where θ Bn > 70°) interplanetary shocks is investigated. This extends the previous study of Neergaard Parker and Zank which focused on the injection problem at quasi-parallel interplanetary shocks. We use observations at 1 AU to construct upstream Maxwellian and κ-distributions that are then diffusively accelerated by the shock, thus yielding the downstream accelerated particle distribution. We compare the theoretical accelerated particle distribution to observations at 1 AU using Advanced Composition Explorer data. We classify our results for quasi-perpendicular shocks into three subcategories: those with ratios of the theoretical spectral index to observed power law of >1, ∼ 1, and <1, and compare the magnetic power spectral density plots of these categories. We find that in general the assumed upstream particle distribution that best fits the energetic particle observations is best represented by a κ-distribution, with κ = 4. The magnetic field fluctuations were representative of quasi-perpendicular shocks and showed no particular bias toward our spectral ratio subcategories. The subcategory with spectral ratio <0.9 yielded the largest injection energies for all groups. In all but two of the cases in this study, there were enough particles in the solar wind thermal core to account for the accelerated distribution, thereby giving a lower limit to the required injection energy needed to diffusively accelerate particles at a quasi-perpendicular interplanetary shock. In the remaining two cases, an additional population of particles was required to match the appropriate amplitude of the spectral index. For these cases, we used a low energy (1-50 keV) v –5 spectrum advocated by Fisk and Gloeckler.

  3. 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.

  4. A SIMPLE EXPERIMENTAL MODEL OF HEAT SHOCK RESPONSE IN RATS

    Directory of Open Access Journals (Sweden)

    Tufi Neder Meyer

    1998-10-01

    Full Text Available Objective: To obtain a simple model for the elicitation of the heat shock response in rats. Design: Laboratory study. Setting: University research laboratories. Sample: Seventy-nine adult male albino rats (weight range 200 g to 570 g. Procedures: Exposure to heat stress by heating animals in a warm bath for 5 min after their rectal temperatures reached 107.60 F (420 C. Liver and lung samples were collected for heat-shock protein 70 (HSP70 detection (Western analysis. Results: Western analysis was positive for HSP70 in the liver and in the lungs of heated animals. There was a temporal correlation between heating and HSP70 detection: it was strongest 1 day after heating and reduced afterwards. No heated animals died. Conclusion: These data show that heating rats in a warm (45o C bath, according to parameters set in this model, elicits efficiently the heat shock response.OBJETIVO: Obter um modelo simples para tentar esclarecer a resposta ao choque térmico em ratos. LOCAL: Laboratório de pesquisa da Universidade. MÉTODO: Amostra: 79 ratos albinos, adultos, entre 200g a 570g. Procedimentos: Exposição ao calor, em banho quente, por 5 minutos, após a temperatura retal chegar a 42 graus centigrados. Biópsias de fígado e pulmão foram obtidas para detectar a proteina 70 (HSP 70, pelo "Western blot". RESULTADOS: As análises foram positivas nos animais aquecidos, com uma correlação entre aquecimento e constatação da HSP 70. Foi mais elevada no primeiro dia e não houve óbitos nos animais aquecidos. CONCLUSÃO: Os ratos aquecidos a 45 graus centígrados respondem eficientemente ao choque térmico.

  5. Identification of Heat Shock Protein families and J-protein types by incorporating Dipeptide Composition into Chou's general PseAAC.

    Science.gov (United States)

    Ahmad, Saeed; Kabir, Muhammad; Hayat, Maqsood

    2015-11-01

    Heat Shock Proteins (HSPs) are the substantial ingredients for cell growth and viability, which are found in all living organisms. HSPs manage the process of folding and unfolding of proteins, the quality of newly synthesized proteins and protecting cellular homeostatic processes from environmental stress. On the basis of functionality, HSPs are categorized into six major families namely: (i) HSP20 or sHSP (ii) HSP40 or J-proteins types (iii) HSP60 or GroEL/ES (iv) HSP70 (v) HSP90 and (vi) HSP100. Identification of HSPs family and sub-family through conventional approaches is expensive and laborious. It is therefore, highly desired to establish an automatic, robust and accurate computational method for prediction of HSPs quickly and reliably. Regard, a computational model is developed for the prediction of HSPs family. In this model, protein sequences are formulated using three discrete methods namely: Split Amino Acid Composition, Pseudo Amino Acid Composition, and Dipeptide Composition. Several learning algorithms are utilized to choice the best one for high throughput computational model. Leave one out test is applied to assess the performance of the proposed model. The empirical results showed that support vector machine achieved quite promising results using Dipeptide Composition feature space. The predicted outcomes of proposed model are 90.7% accuracy for HSPs dataset and 97.04% accuracy for J-protein types, which are higher than existing methods in the literature so far. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. 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.

  7. 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.

  8. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  9. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    International Nuclear Information System (INIS)

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-01-01

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR γ ) and CCAAT element binding protein α (C/EBP α ), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins

  10. Small heat shock proteins protect against α-synuclein-induced toxicity and aggregation

    International Nuclear Information System (INIS)

    Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.; Liu Fang; Nguyen, Paul; Rochet, Jean-Christophe; Hyman, Bradley T.; McLean, Pamela J.

    2006-01-01

    Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). α-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and αB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are ∼2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by ∼80% in a culture model while αB-crystallin reduces toxicity by ∼20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model

  11. Heat-shock proteins in stromal joint tissues: innocent bystanders or disease-initiating proteins?

    Science.gov (United States)

    Lambrecht, Stijn; Juchtmans, Nele; Elewaut, Dirk

    2014-02-01

    Heat-shock proteins (HSPs) are molecular chaperones that are highly conserved between species. In recent decades it has become clear that these proteins play an important role in the pathogenesis of inflammatory and degenerative joint diseases by (dys)regulating the immune system and by direct effects on the stromal tissues of the joint. In this review we discuss current insights into the expression pattern of HSPs in connective tissues, the direct biological role of HSPs in stromal tissues and the potential clinical applications.

  12. 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.

  13. Heat shock protein 90 (Hsp90) chaperone complex. A molecular target for enhancement of thermosensitivity and radiosensitivity

    International Nuclear Information System (INIS)

    Akimoto, Tetsuo; Nonaka, Tetsuo; Kitamoto, Yoshizumi; Sakurai, Hideyuki

    2002-01-01

    Heat shock protein 90 (Hsp90) is a highly conserved heat shock protein in animal and plants, and exists abundantly in the cytoplasm in unstressed condition, accounting for 1-2% in cytoplasmic proteins. Main difference of Hsp90 from other Hsps are its substrate that Hsp90 binds to. These substrates include various signal transduction proteins, kinase, steroid receptors and transcription factors, therefore, Hsp90 plays a key role in maintaining cellular signal transduction networks. Many chaperoned proteins (client proteins) of Hsp90 are associated with cellular proliferation or malignant transformation, thus Hsp90 chaperone complex has been focused as targets for cancer therapy. Among the client proteins, there are several molecules that have been defined as targets or factors for determination or enhancement of radiosensitivity or thermosensitivity. Thus, it is easily speculated that Hsp90 chaperone complex inhibitors that disrupt association of Hsp90 and client protein in combination with radiation or/and heat has potential effect on enhancement of radiosensitivity or thermosensitivity. In this paper, possible mechanisms in enhancing radiosensitivity or thermosensitivity according to the client proteins will be summarized. (author)

  14. Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs.

    Science.gov (United States)

    Sundarraj, Kiruthika; Raghunath, Azhwar; Panneerselvam, Lakshmikanthan; Perumal, Ekambaram

    2017-02-15

    With increased industrial utilization of iron oxide nanoparticles (Fe 2 O 3 -NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe 2 O 3 -NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe 2 O 3 -NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe 2 O 3 -NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe 2 O 3 -NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe 2 O 3 -NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe 2 O 3 -NPs could be an environmental risk factor for reproductive disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. The Potential Coordination of the Heat-Shock Proteins and Antioxidant Enzyme Genes of Aphidius gifuensis in Response to Thermal Stress

    Science.gov (United States)

    Kang, Zhi-Wei; Liu, Fang-Hua; Liu, Xiang; Yu, Wen-Bo; Tan, Xiao-Ling; Zhang, Shi-Ze; Tian, Hong-Gang; Liu, Tong-Xian

    2017-01-01

    Aphidius gifuensis is one of the most important aphid natural enemies and has been successfully used to control Myzys persicae and other aphid species. High temperature in summer is one of the key barriers for the application of A. gifuensis in the field and greenhouse. In this work, we investigated the biological performance of A. gifuensis and the response of heat-shock proteins and antioxidant enzymes under high temperature. The results showed that A. gifuensis could not survive at 40°C and female exhibited a higher survival in 35°C. Furthermore, the short term exposure to high temperature negatively affected the performance of A. gifuensis especially parasitism efficiency. Under short-term heating, the expression of AgifsHSP, Agifl(2)efl, AgifHSP70, AgifHSP70-4 and AgifHSP90 showed an increased trend, whereas AgifHSP10 initially increased and then decreased. In 35°C, the expressions of Agifl(2)efl, AgifHSP70-4 and AgifHSP90 in female were higher than those in male, whereas the expression of AgifHSP70 exhibited an opposite trend. Besides the HSPs, we also quantified the expression levels of 11 antioxidant enzyme genes: AgifPOD, AgifSOD1, AgifSOD2, AgifSOD3, AgifCAT1, AgifCAT2, AgifGST1, AgifGST2, AgifGST3, AgifGST4 and AgifGST5. We found that the sex-specific expression of AgifSOD2, AgifSOD3, AgifPOD, AgifGST1 and AgifGST3 were highly consistent with sex-specific heat shock survival rates at 35°C. Furthermore, when the temperature was above 30°C, the activities of GST, SOD, CAT and POD were significantly increased; however, there was no significant difference of the CAT activity between the male and female at 35°C. Collectively, all of these results suggested that the protection of thermal damage is coordinated by HSPs and antioxidant enzymes in A. gifuensis. Based on the heat tolerance abilities of many aphid natural enemies, we also discussed an integrated application strategy of many aphid enemies in summer. PMID:29234290

  16. The heat-shock, or HSF1-mediated proteotoxic stress, response in cancer: from proteomic stability to oncogenesis.

    Science.gov (United States)

    Dai, Chengkai

    2018-01-19

    The heat-shock, or HSF1-mediated proteotoxic stress, response (HSR/HPSR) is characterized by induction of heat-shock proteins (HSPs). As molecular chaperones, HSPs facilitate the folding, assembly, transportation and degradation of other proteins. In mammals, heat shock factor 1 (HSF1) is the master regulator of this ancient transcriptional programme. Upon proteotoxic insults, the HSR/HPSR is essential to proteome homeostasis, or proteostasis, thereby resisting stress and antagonizing protein misfolding diseases and ageing. Contrasting with these benefits, an unexpected pro-oncogenic role of the HSR/HPSR is unfolding. Whereas HSF1 remains latent in primary cells without stress, it becomes constitutively activated within malignant cells, rendering them addicted to HSF1 for their growth and survival. Highlighting the HSR/HPSR as an integral component of the oncogenic network, several key pathways governing HSF1 activation by environmental stressors are causally implicated in malignancy. Importantly, HSF1 impacts the cancer proteome systemically. By suppressing tumour-suppressive amyloidogenesis, HSF1 preserves cancer proteostasis to support the malignant state, both providing insight into how HSF1 enables tumorigenesis and suggesting disruption of cancer proteostasis as a therapeutic strategy. This review provides an overview of the role of HSF1 in oncogenesis, mechanisms underlying its constitutive activation within cancer cells and its pro-oncogenic action, as well as potential HSF1-targeting strategies.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'. © 2017 The Author(s).

  17. 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.

  18. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    Science.gov (United States)

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

  19. 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.

  20. Cry1Ab treatment has no effects on viability of cultured porcine intestinal cells, but triggers Hsp70 expression.

    Directory of Open Access Journals (Sweden)

    Angelika Bondzio

    Full Text Available In vitro testing can contribute to reduce the risk that the use of genetically modified (GM crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2 as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.

  1. Molecular cloning, phylogenetic analysis and heat shock response of Babesia gibsoni heat shock protein 90.

    Science.gov (United States)

    Yamasaki, Masahiro; Tsuboi, Yoshihiro; Taniyama, Yusuke; Uchida, Naohiro; Sato, Reeko; Nakamura, Kensuke; Ohta, Hiroshi; Takiguchi, Mitsuyoshi

    2016-09-01

    The Babesia gibsoni heat shock protein 90 (BgHSP90) gene was cloned and sequenced. The length of the gene was 2,610 bp with two introns. This gene was amplified from cDNA corresponding to full length coding sequence (CDS) with an open reading frame of 2,148 bp. A phylogenetic analysis of the CDS of HSP90 gene showed that B. gibsoni was most closely related to B. bovis and Babesia sp. BQ1/Lintan and lies within a phylogenetic cluster of protozoa. Moreover, mRNA transcription profile for BgHSP90 exposed to high temperature were examined by quantitative real-time reverse transcription-polymerase chain reaction. BgHSP90 levels were elevated when the parasites were incubated at 43°C for 1 hr.

  2. Possible Contribution of Zerumbone-Induced Proteo-Stress to Its Anti-Inflammatory Functions via the Activation of Heat Shock Factor 1.

    Directory of Open Access Journals (Sweden)

    Yoko Igarashi

    Full Text Available Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1 plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.

  3. 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.

  4. Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

    International Nuclear Information System (INIS)

    Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

    1987-01-01

    The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

  5. 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.

  6. Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas.

    Science.gov (United States)

    MacRae, T H

    2000-06-01

    Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.

  7. Extracellular small heat shock proteins: exosomal biogenesis and function.

    Science.gov (United States)

    Reddy, V Sudhakar; Madala, Satish K; Trinath, Jamma; Reddy, G Bhanuprakash

    2018-05-01

    Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.

  8. Expression of small heat shock proteins from pea seedlings under gravity altered conditions

    Science.gov (United States)

    Talalaev, Alexandr S.

    2005-08-01

    A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

  9. Responses of antioxidant enzymes and heat shock proteins in drosophila to treatment with a pesticide mixture

    Directory of Open Access Journals (Sweden)

    Doganlar Oguzhan

    2015-01-01

    Full Text Available The effects of a mixture of seven pesticides were examined on the expression of antioxidant enzymes, Mn superoxide dismutase (Mn-SOD, catalase (CAT, glutathione synthetase (GS, and heat shock proteins (HSP 26, 60, 70 and 83 in adult fruit flies (Drosophila melanogaster Oregon R. The flies were reared under controlled conditions on artificial diets and treated with a mixture of seven pesticides (molinate, thiobencarb, linuron, phorate, primiphos-methyl, fenvalerate and lambda-cyhalothrin commonly found in water, at concentrations of 0.1, 0.5 and 1 parts per billion (ppb for 1 and 5 days. Quantitative real-time PCR (qRT-PCR analysis of Mn-SOD, CAT and GS expression revealed that the analyzed markers responded significantly to pesticide-induced oxidative stress, in particular on the 5th day of treatment. On the 1st day of treatment, the relative expression of HSP26 and HSP60 genes increased only after exposure to the highest concentrations of pesticides, whereas HSP70 and HSP83 expression increased after exposure to 0.5 and 1 ppb. After five days of treatment, the expression of all HSP genes was increased after exposure to all pesticide concentrations. A positive correlation was determined between the relative expression levels of some HSPs (except HSP60, and antioxidant genes. The observed changes in antioxidant enzyme and HSP mRNA levels in D. melanogaster suggest that the permissible limits of pesticide concentrations for clean drinking water outlined in the regulations of several countries are potentially cytotoxic. The presented findings lend support for reevaluation of these limits.

  10. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  11. 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.

  12. The change in heat shock protein expression in avermectin induced neurotoxicity of the pigeon (Columba livia) both in vivo and in vitro.

    Science.gov (United States)

    Li, Ming; Wang, Xian-Song; Xu, Feng-Ping; Liu, Shuang; Xu, Shi-Wen; Li, Shu

    2014-12-01

    The expression of heat shock proteins (Hsps) commonly increases to provide neuroprotection when brain tissues are under stress conditions. Residues of avermectins (AVMs) have neurotoxic effects on a number of non-target organisms. The aim of this study was to investigate the effects of AVM exposure on the expression levels of Hsp 60, Hsp 70 and Hsp 90 for pigeon (Columba livia) neurons both in vivo and in vitro. The results showed that in general, the mRNA and protein levels of Hsps were increased in treated groups relative to control groups after AVM exposure for 30d, 60d and 90d in the cerebrum, cerebellum and optic lobe in vivo. However, AVM exposure had no significant effects on the transcription expression of Hsps for 90d in the optic lobe and decreased the translation expression of Hsps significantly for 90d in the optic lobe. In vitro, the LC50 of avermectin for King pigeon neurons is between 15μgL(-1) and 20μgL(-1). Following AVM (2.5-20μgL(-1)) exposure, the mRNA expression of the 3 Hsps was up-regulated to different degrees. Compared with the control groups, a significant decrease, a remarkable increase and a non-significant change was found in the protein expression of Hsp 60, Hsp 70 and Hsp 90 separately following AVM (2.5-20μgL(-1)) exposure. Based on these results, we conclude that AVM exposure can induce a protective stress response in pigeons by means of promoting the mRNA and protein expression of Hsps under in vivo and in vitro conditions, thus easing the neurotoxic effects of AVM to some extent. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

    DEFF Research Database (Denmark)

    Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per

    2014-01-01

    respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

  14. 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.

  15. 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.

  16. Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers

    Science.gov (United States)

    Massip-Salcedo, Marta; Casillas-Ramirez, Araní; Franco-Gou, Rosah; Bartrons, Ramón; Ben Mosbah, Ismail; Serafin, Anna; Roselló-Catafau, Joan; Peralta, Carmen

    2006-01-01

    Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery. PMID:16651615

  17. The induced expression of heat shock proteins as a part of the early cellular response to gamma radiation

    International Nuclear Information System (INIS)

    Stankova, K.; Ivanova, K.; Georgieva, R.; Rupova, I.; Boteva, R.

    2008-01-01

    A variety of stressful stimuli including gamma radiation can induce increase in the synthesis of heat shock proteins (Hsp). This family of molecular chaperones includes members with molecular masses ranging from 10 to 150 kDa and has been identified in all organisms from bacteria to humans. Hsp70 chaperones are very important. The present study aimed to characterize the radiation-induced changes in Hsp70 synthesis in human lymphocytes as a part of the early cellular response to gamma irradiation. The expression of Hsp70 was determined with Western blot and the radiation-induced apoptotic changes were registered by staining with fluorescent dyes. Part of the experiments were performed in the presence of the organic solvent DMSO. At low concentrations this reagent shows antioxidant activity and can reduce the level of the radiation-induced oxidant stress which determines the predominant biological effects of the ionizing radiation. Irradiation with 0.5 to 8 Gy caused statistically significant increase in the synthesis of Hsp70 which was strongest after irradiation with 4 Gy. In the range 0.5-2 Gy the enhancement of the radiation-induced synthesis of Hsp70 reached 60%. Our experimental results characterize changes in the Hsp70 synthesis after gamma irradiation as a part of the early cellular stress response in lymphocytes. (authors)

  18. 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

  19. The effect of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress.

    Science.gov (United States)

    Sesay, Daniella Fatmata; Habte-Tsion, Habte-Michael; Zhou, Qunlan; Ren, Mingchun; Xie, Jun; Liu, Bo; Chen, Ruli; Pan, Liangkun

    2017-08-01

    The effects of dietary folic acid on biochemical parameters and gene expression of three heat shock proteins (HSPs) of blunt snout bream (Megalobrama amblycephala) fingerling under acute high temperature stress. Six dietary folic acid groups (0.0, 0.5, 1.0, 2.0, 5.0, and 10.0) mg/kg diets were designed and assigned into 18 tanks in three replicates each (300 l/tank) and were administered for 10 weeks in a re-circulated water system. The fingerlings with an initial weight of 27.0 ± 0.03 g were fed with their respective diets four times daily. At the end of the experiment, samples were collected before challenge, 0, 24, 72 h, and 7 days. Serum total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cortisol, glucose, complement C3 (C3), complement C4 (C4, immunoglobulin M (IgM) hepatic superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and the expression of heat shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) were studied. The results showed that fish fed with dietary folic acid between 1.0, 2.0, and 5.0 mg/kg significantly (P stress, 0, 24, 72 h, and 7 days significantly (P  0.05) of the three HSPs. However, there were statistical significant interactive effect between dietary folic acid inclusion level and temperature duration on serum C3 and C4 (P  0.05). The present results indicate that supplementation of basal diet from 1.0 mg/kg; 2.0 and 5.0 mg/kg can enhance acute high temperature resistance ability in M. amblycephala fingerling to some degree and improve physiological response, immune and antioxidant capacities, and expression level of three HSPs.

  20. 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.

  1. Small heat shock proteins can release light dependence of tobacco seed during germination.

    Science.gov (United States)

    Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia; Hong, Choo Bong

    2015-03-01

    Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. © 2015 American Society of Plant Biologists. All Rights Reserved.

  2. Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

    Science.gov (United States)

    Salimi, Leila; Rahbarghazi, Reza; Jafarian, Vahab; Biray Avci, Çıgır; Goker Bagca, Bakiye; Pinar Ozates, Neslihan; Khaksar, Majid; Nourazarian, Alireza

    2018-01-18

    In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (p control vs 70 mM group  cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition. © 2018 Wiley Periodicals, Inc.

  3. Heat shock protein 70 and tumor-infiltrating NK cells as prognostic indicators for patients with squamous cell carcinoma of the head and neck after radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

    Science.gov (United States)

    Stangl, Stefan; Tontcheva, Nikoletta; Sievert, Wolfgang; Shevtsov, Maxim; Niu, Minli; Schmid, Thomas E; Pigorsch, Steffi; Combs, Stephanie E; Haller, Bernhard; Balermpas, Panagiotis; Rödel, Franz; Rödel, Claus; Fokas, Emmanouil; Krause, Mechthild; Linge, Annett; Lohaus, Fabian; Baumann, Michael; Tinhofer, Inge; Budach, Volker; Stuschke, Martin; Grosu, Anca-Ligia; Abdollahi, Amir; Debus, Jürgen; Belka, Claus; Maihöfer, Cornelius; Mönnich, David; Zips, Daniel; Multhoff, Gabriele

    2018-05-01

    Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre-activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor-infiltrating CD56 + NK cells in formalin-fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin-based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3-4) showed significantly decreased overall survival (OS; p = 0.008), local progression-free survival (LPFS; p = 0.034) and distant metastases-free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0-2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16- (p = 0.001), p53- (p = 0.0003) and HPV16 DNA-negative (p = 0.001) tumors. The absence or low numbers of tumor-infiltrating CD56 + NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor-infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor-infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  4. Distinct radioprotective activities of major heat shock proteins in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Kabakov, Alexander; Malyutina, Yana; Kudryavtsev, Vladimir

    2008-01-01

    Full text: Several years ago we have suggested that heat shock proteins (Hsps) can be involved in cellular and tissue mechanisms of protection from ionizing radiation. At present, the accumulated experimental data do allow us to characterize three major mammalian Hsps, Hsp70, Hsp27 and Hsp90, as specific endogenous radioprotectors which are able to prevent or minimize cell death resulting from the radiation exposure. It follows from the many findings that the radioprotective effect of these Hsps is particularly manifested in their ability to attenuate apoptosis in various normal and tumor cells irradiated in vivo or in vitro. The obtained data already enable to suggest three main mechanisms of the radioprotection conferred by the excess Hsps: 1) Modulation of the intracellular signaling so that the apoptotic signal transduction is blocked, whereas the 'cell survival' signal transduction is stimulated; 2) Suppression of the radiation-associated free radical generation and apoptosis induced by reactive oxygen species (ROS); 3) Attenuation of the genotoxic impact of ionizing radiation. The latter suggested mechanism seems particularly intriguing and implies that the excess Hsps can somehow contribute to protection/repair of genomic DNA from radiation-induced damage. According to our recent results, Hsp90 is indeed involved in the post-irradiation repair of nuclear DNA, while excess Hsp70 can beneficially affect the p53-mediated DNA damage response in irradiated cells to ensure their long-term survival and recovery. As for Hsp27, we found that its accumulation in target cells increases their radioresistance by enhancing the irradiation-responsive activation of anti apoptotic pathways. While the Hsp70 and Hsp27 seem to perform different functions in irradiated cells, the synergistic enhancement of radioprotection was clearly observed in the cells enriched by the both the Hsps. In vivo, such radioprotective activities of the major mammalian Hsps may play a role in

  5. 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

  6. 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)

  7. Forkhead Box M1 Is Regulated by Heat Shock Factor 1 and Promotes Glioma Cells Survival under Heat Shock Stress*

    Science.gov (United States)

    Dai, Bingbing; Gong, Aihua; Jing, Zhitao; Aldape, Kenneth D.; Kang, Shin-Hyuk; Sawaya, Raymond; Huang, Suyun

    2013-01-01

    The forkhead box M1 (FoxM1) is a key transcription factor regulating multiple aspects of cell biology. Prior studies have shown that FoxM1 is overexpressed in a variety of human tumors, including brain tumor, and plays a critical role in cancer development and progression. In this study we found that FoxM1 was up-regulated by heat shock factor 1 (HSF1) under heat shock stress condition in multiple cell lines. Knockdown of HSF1 with HSF1 siRNA or inhibition of HSF1 with a HSF1 inhibitor abrogated heat shock-induced expression of FoxM1. Genetic deletion of HSF1 in mouse embryo fibroblast cells also abolished heat shock stress-induced FoxM1 expression. Moreover, we showed that HSF1 directly bound to FoxM1 promoter and increased FoxM1 promoter activity. Furthermore, we demonstrated that FoxM1 was required for the G2-M phase progression through regulating Cdc2, Cdc20, and Cdc25B under a mild heat shock stress but enhanced cell survival under lethal heat shock stress condition. Finally, in human glioblastoma specimens, FoxM1 overexpression correlated with elevated HSF1 expression. Our results indicate that FoxM1 is regulated by HSF1 and is critical for HSF1-mediated heat shock response. We demonstrated a novel mechanism of stress resistance controlled by HSF1 and a new HSF-FoxM1 connection that mediates cellular thermotolerance. PMID:23192351

  8. Heat shock protein-peptide complex-96 (Vitespen for the treatment of cancer

    Directory of Open Access Journals (Sweden)

    Robert J. Amato

    2011-12-01

    Full Text Available Heat shock proteins (HSPs are the most abundant and ubiquitous soluble intracellular proteins. Members of the HSP family bind peptides, they include antigenic peptides generated within cells. HSPs also interact with antigen-presenting cells (APCs through CD91 and other receptors, eliciting a cascade of events that includes re-presentation of HSP-chaperoned peptides by major histocompatability complex (MHC, translocation of nuclear factorkappaB (NFkB into the nuclei, and maturation of dendritic cells (DCs. These consequences point to a key role of heat shock proteins in fundamental immunological phenomena such as activation of APCs, indirect presentation (or crosspriming of antigenic peptides, and chaperoning of peptides during antigen presentation. The properties of HSPs also allow them to be used for immunotherapy of cancers and infections in novel ways. This paper reviews the development and clinical trial progress of vitespen, an HSP peptide complex vaccine based on tumor-derived glycoprotein 96.

  9. 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.

  10. Microarray-based screening of heat shock protein inhibitors.

    Science.gov (United States)

    Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

    2014-06-20

    Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1

    DEFF Research Database (Denmark)

    Wallrodt, Inke; Jelsbak, Lotte; Thomsen, Line E.

    2014-01-01

    The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene......IV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA....

  12. 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

  13. Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

    Science.gov (United States)

    Uma, Sheri; Thulasiraman, Vanitha; Matts, Robert L.

    1999-01-01

    The heme-regulated kinase of the α subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI. PMID:10454533

  14. Heat-shock responses in two leguminous plants: a comparative study.

    Science.gov (United States)

    Ortiz, C; Cardemil, L

    2001-08-01

    Relative growth rates, basal and acclimated thermotolerance, membrane damage, fluorescence emission, and relative levels of free and conjugated ubiquitin and HSP70 were compared after 2 h of treatment at different temperatures between Prosopis chilensis and Glycine max (soybean), cv. McCall, to evaluate if the thermotolerance of these two plants was related to levels of accumulation of heat shock proteins. Seedlings of P. chilensis germinated at 25 degrees C and at 35 degrees C and grown at temperatures above germination temperature showed higher relative growth than soybean seedlings treated under the same conditions. The lethal temperature of both species was 50 degrees C after germination at 25 degrees C. However, they were able to grow at 50 degrees C after germination at 35 degrees C. Membrane damage determinations in leaves showed that P. chilensis has an LT(50) 6 degrees C higher than that of soybean. There were no differences in the quantum yield of photosynthesis (F(v)/F(m)), between both plants when the temperatures were raised. P. chilensis showed higher relative levels of free ubiquitin, conjugated ubiquitin and HSP70 than soybean seedlings when the temperatures were raised. Time-course studies of accumulation of these proteins performed at 40 degrees C showed that the relative accumulation rates of ubiquitin, conjugated ubiquitin and HSP70 were higher in P. chilensis than in soybean. In both plants, free ubiquitin decreased during the first 5 min and increased after 30 min of heat shock, conjugated ubiquitin increased after 30 min and HSP70 began to increase dramatically after 20 min of heat shock. From these data it is concluded that P. chilensis is more tolerant to acute heat stress than soybean.

  15. Synergistic skin heat shock protein expression in response to combined laser treatment with a diode laser and ablative fractional lasers.

    Science.gov (United States)

    Paasch, Uwe; Sonja, Grunewald; Haedersdal, Merete

    2014-06-01

    Diode laser-based skin heating has been shown to minimise scars by interfering with wound healing responses through the induction of heat shock proteins (HSP). HSP are also induced after ablative fractional laser (AFXL) wound healing. AFXL itself is highly recommended for scar treatment. Therefore, the sequential combination of both modalities may produce superior outcomes. The aim of this study was to examine the pretreatment effects of a diode laser before AFXL on wound healing responses in terms of HSP up-regulation in an in vitro model. Immediate responses and responses on days 1, 3 or 6 post-procedure were studied in an in vitro porcine skin model (n = 240). Untreated samples served as control. Immunohistochemical investigation (Hsp70) was performed in all untreated controls, diode laser-, AFXL-, and in diode laser + AFXL-treated samples. Hsp70 was shown to be up-regulated by all interventions between days 1 and 6 after interventions. The largest effect was caused by the combination of a diode laser and an AFXL procedure. Diode laser exposure induces a skin HSP response that can be further enhanced by sequential AFXL treatment. Clinical studies are necessary to investigate the dose response of HSP on scar formation and refine suitable laser exposure settings.

  16. Mapping the Interactome of a Major Mammalian Endoplasmic Reticulum Heat Shock Protein 90.

    Directory of Open Access Journals (Sweden)

    Feng Hong

    Full Text Available Up to 10% of cytosolic proteins are dependent on the mammalian heat shock protein 90 (HSP90 for folding. However, the interactors of its endoplasmic reticulum (ER paralogue (gp96, Grp94 and HSP90b1 has not been systematically identified. By combining genetic and biochemical approaches, we have comprehensively mapped the interactome of gp96 in macrophages and B cells. A total of 511 proteins were reduced in gp96 knockdown cells, compared to levels observed in wild type cells. By immunoprecipitation, we found that 201 proteins associated with gp96. Gene Ontology analysis indicated that these proteins are involved in metabolism, transport, translation, protein folding, development, localization, response to stress and cellular component biogenesis. While known gp96 clients such as integrins, Toll-like receptors (TLRs and Wnt co-receptor LRP6, were confirmed, cell surface HSP receptor CD91, TLR4 pathway protein CD180, WDR1, GANAB and CAPZB were identified as potentially novel substrates of gp96. Taken together, our study establishes gp96 as a critical chaperone to integrate innate immunity, Wnt signaling and organ development.

  17. Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

    International Nuclear Information System (INIS)

    Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

    2003-01-01

    Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

  18. Neurofilaments Function as Shock Absorbers: Compression Response Arising from Disordered Proteins

    Science.gov (United States)

    Kornreich, Micha; Malka-Gibor, Eti; Zuker, Ben; Laser-Azogui, Adi; Beck, Roy

    2016-09-01

    What can cells gain by using disordered, rather than folded, proteins in the architecture of their skeleton? Disordered proteins take multiple coexisting conformations, and often contain segments which act as random-walk-shaped polymers. Using x-ray scattering we measure the compression response of disordered protein hydrogels, which are the main stress-responsive component of neuron cells. We find that at high compression their mechanics are dominated by gaslike steric and ionic repulsions. At low compression, specific attractive interactions dominate. This is demonstrated by the considerable hydrogel expansion induced by the truncation of critical short protein segments. Accordingly, the floppy disordered proteins form a weakly cross-bridged hydrogel, and act as shock absorbers that sustain large deformations without failure.

  19. Interplanetary shock phenomena beyond 1 AU

    International Nuclear Information System (INIS)

    Smith, E.J.

    1985-01-01

    Attention is given to spatial dependences exhibited by spacecraft measurements obtained between 1 and 30 AU, together with temporal variations occurring between solar activity cycle maxima and minima. At 1-3 AU radial distances, shocks develop in association with the corotating solar wind streams characterizing solar minimum and accelerate solar wind evolution with distance while heating the solar wind and generating waves and turbulence. At solar maximum, shocks are observed more frequently at 1 AU but still in association with transient solar events; acceleration leading to energetic storm particles is observed both within and beyond 1 AU. The superimposed effect of large numbers of intense shocks may be responsible for the solar cycle modulation of galactic cosmic rays. 77 references

  20. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  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. [Suppressive Effects of Extract of Cedar Wood on Heat-induced Expression of Cellular Heat Shock Protein].

    Science.gov (United States)

    Miyakoshi, Junji; Matsubara, Eri; Narita, Eijiro; Koyama, Shin; Shimizu, Yoko; Kawai, Shuichi

    2018-01-01

     In recent years, highly antimicrobial properties of cedar heartwood essential oil against the wood-rotting fungi and pathogenic fungi have been reported in several papers. Antimicrobial properties against oral bacteria by hinokitiol contained in Thujopsis have been also extensively studied. The relation of naturally derived components and human immune system has been studied in some previous papers. In the present study, we focused on Japanese cedar, which has the widest artificial afforestation site in the country among various tree species. Extract oil was obtained from mixture of sapwood and heartwood of about 40-year cedar grown in Oguni, Kumamoto, Japan. We examined the influence of extract components from Japanese cedar woods on the expression of heat shock protein 70 (Hsp70) during heating, and on the micronucleus formation induced by the treatment of bleomycin as a DNA damaging agent. Cell lines used in this study were human fetal glial cells (SVGp12) and human glioma cells (MO54). Remarkable suppression of the Hsp70 expression induced by heating at 43°C was detected by the treatment of cedar extract in both SVGp12 and MO54 cells. We also found that cedar extract had an inhibitory tendency to reduce the micronucleus formation induced by bleomycin. From these results, the extract components from Japanese cedar woods would have an inhibitory effect of the stress response as a suppression of the heat-induced Hsp70 expression, and might have a reductive effect on carcinogenicity.

  3. 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.

  4. DIFFERENTIAL RESPONSE OF HEAT SHOCK PROTEINS TO UPHILL AND DOWNHILL EXERCISE IN HEART, SKELETAL MUSCLE, LUNG AND KIDNEY TISSUES

    Directory of Open Access Journals (Sweden)

    Pablo C. B. Lollo

    2013-09-01

    Full Text Available Running on a horizontal plane is known to increase the concentration of the stress biomarker heat-shock protein (HSP, but no comparison of the expression of HSP70 has yet been established between the uphill (predominantly concentric and downhill (predominantly eccentric muscle contractions exercise. The objective of the study was to investigate the relationships between eccentric and concentric contractions on the HSP70 response of the lung, kidney, gastrocnemius, soleus and heart. Twenty-four male Wistar weanling rats were divided into four groups: non-exercised and three different grades of treadmill exercise groups: horizontal, uphill (+7% and downhill (-7% of inclination. At the optimal time-point of six hours after the exercise, serum uric acid, creatine kinase (CK and lactate dehydrogenase (LDH were determined by standard methods and HSP70 by the Western blot analysis. HSP70 responds differently to different types of running. For kidney, heart, soleus and gastrocnemius, the HSP70 expression increased, 230, 180, 150 and 120% respectively of the reference (horizontal. When the contraction was concentric (uphill and compared to downhill the increase in response of HSP70 was greater in 80% for kidney, 75% for gastrocnemius, 60% for soleus and 280% for the heart. Uric acid was about 50% higher (0.64 ± 0.03 mg·dL-1 in the uphill group as compared to the horizontal or downhill groups. Similarly, the activities of serum CK and LDH were both 100% greater for both the uphill and downhill groups as compared to the horizontal group (2383 ± 253 and 647.00 ± 73 U/L, respectively. The responsiveness of HSP70 appeared to be quite different depending on the type of tissue, suggesting that the impact of exercise was not restricted to the muscles, but extended to the kidney tissue. The uphill exercise increases HSP70 beyond the eccentric type and the horizontal running was a lower HSP70 responsive stimulus

  5. 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.

  6. Table 1.xlsx

    Indian Academy of Sciences (India)

    Sandra Feijoo Bandin

    60 Kda heat shock protein, mitochondrial. GRP75_MOUSE Stress-70 protein, mitochondrial. PHB_MOUSE. Prohibitin. Biological process. Metabolic process. Glycolysis. Lipid metabolism. Tricarboxylic acid cycle. Respiratory electron transport chain. Muscle contraction. Cellular component organization. Transport.

  7. Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDa Vibrio cholerae heat-shock protein VcHsp31

    International Nuclear Information System (INIS)

    Das, Samir; Dey, Sanjay; Roy, Trina; Sen, Udayaditya

    2011-01-01

    A heat-shock protein from V. cholerae (VcHsp31) has been cloned, expressed, purified and crystallized. Crystals of VcHsp31 belonged to a monoclinic space group and diffracted to 1.9 Å resolution. The Gram-negative bacterium Vibrio cholerae, which is responsible for the diarrhoeal disease cholera in humans, induces the expression of numerous heat-shock genes. VcHsp31 is a 31 kDa putative heat-shock protein that belongs to the DJ-1/PfpI superfamily, functioning as both a chaperone and a protease. VcHsp31 has been cloned, overexpressed and purified by Ni 2+ –NTA affinity chromatography followed by gel filtration. Crystals of VcHsp31 were grown in the presence of PEG 6000 and MPD; they belonged to space group P2 1 and diffracted to 1.9 Å resolution. Assuming the presence of six molecules in the asymmetric unit, the Matthews coefficient was estimated to be 1.97 Å 3 Da −1 , corresponding to a solvent content of 37.4%

  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. Molecular characterization and expression analysis of a heat shock protein 90 gene from disk abalone (Haliotis discus).

    Science.gov (United States)

    Wang, Ning; Whang, Ilson; Lee, Jae-Seong; Lee, Jehee

    2011-06-01

    Heat shock protein 90s (hsp90s) are chaperones that contribute to the proper folding of cellular proteins and help animals cope with the cellular protein damages in stress conditions. In this study, an hsp90 gene was isolated from disc abalone (Haliotis discus). The complete nucleotide sequence of the hsp90 gene contains an open reading frame of 2,184 base pairs, encoding an 84 kDa protein. Disk abalone hsp90 shares high sequence similarity with other hsp90 family proteins. Although the phylogenetic analysis did not classify it into the hsp90α group, the inductivity of this gene was confirmed by heat shock and lipopolysaccharide (LPS) challenge test. Disk abalone hsp90 gene displayed a rapid and reversible induction response to both an exposure of typical heat shock and the LPS challenge. Once given the sublethal heat shock treatment, the transcription of disk abalone hsp90 gene was significantly up-regulated. With a recovery of 12 h, the transcription of disk abalone hsp90 gene gradually attenuated to the control level. These observations reflected the feedback regulation of abalone heat shock responses faithfully. In response to LPS challenge, the transcription of disk abalone hsp90 gene was significantly increased within 2 h and it approached maximum induction at 4 h later and recovered finally the reference level in 24 h. Take all together, the cloning and expression analysis of disk abalone hsp90 gene provided useful molecular information of abalone responses in stress conditions and potential ways to monitor the chronic stressors in abalone culture environments and diagnose the animal health status.

  10. 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

  11. 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

  12. 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.

  13. Heat shock factor-1 intertwines insulin/IGF-1, TGF-β and cGMP signaling to control development and aging

    Directory of Open Access Journals (Sweden)

    Barna János

    2012-11-01

    Full Text Available Abstract Background Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1 functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones (also called heat shock proteins that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown. Results We demonstrate here that in Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-β (transforming growth factor-beta ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1 signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-β and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-β pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation. Conclusion Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1.

  14. The role of the molecular chaperone heat shock protein A2 (HSPA2 in regulating human sperm-egg recognition

    Directory of Open Access Journals (Sweden)

    Brett Nixon

    2015-01-01

    Full Text Available One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI, recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2, as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.

  15. 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.

  16. 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...

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  19. Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

    International Nuclear Information System (INIS)

    Gailit, James

    1990-01-01

    Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Drinking a hot blood meal elicits a protective heat shock response in mosquitoes.

    Science.gov (United States)

    Benoit, Joshua B; Lopez-Martinez, Giancarlo; Patrick, Kevin R; Phillips, Zachary P; Krause, Tyler B; Denlinger, David L

    2011-05-10

    The mosquito's body temperature increases dramatically when it takes a blood meal from a warm-blooded, vertebrate host. By using the yellow fever mosquito, Aedes aegypti, we demonstrate that this boost in temperature following a blood meal prompts the synthesis of heat shock protein 70 (Hsp70). This response, elicited by the temperature of the blood meal, is most robust in the mosquito's midgut. When RNA interference is used to suppress expression of hsp70, protein digestion of the blood meal is impaired, leading to production of fewer eggs. We propose that Hsp70 protects the mosquito midgut from the temperature stress incurred by drinking a hot blood meal. Similar increases in hsp70 were documented immediately after blood feeding in two other mosquitoes (Culex pipiens and Anopheles gambiae) and the bed bug, Cimex lectularius, suggesting that this is a common protective response in blood-feeding arthropods.

  5. Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis

    DEFF Research Database (Denmark)

    Martin-Ventura, Jose Luis; Duran, Mari Carmen; Blanco-Colio, Luis Miguel

    2004-01-01

    -DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27). Surprisingly, compared with control arteries, HSP27 release was drastically decreased in atherosclerotic plaques and barely detectable in complicated plaque supernatants. HSP27 was expressed primarily...... by intact vascular cells of normal arteries and carotid plaques (immunohistochemistry). Plasma detection of soluble HSP27 showed that circulating HSP27 levels are significantly decreased in the blood of patients with carotid stenosis relative to healthy subjects (0.19 [0.1 to 1.95] versus 83 [71.8 to 87...

  6. The role of heat shock protein (HSP as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture

    Directory of Open Access Journals (Sweden)

    Sri Wigati Mardi Mulyani

    2014-03-01

    Full Text Available Background: The concept of stem cell therapy is one of the new hope as a medical therapy on salivary gland defect. However, the lack of viability of the transplanted stem cells survival rate led to the decrease of effectiveness of stem cell therapy. The underlying assumption in the decrease of viability and function of stem cells is an increase of apoptosis incidence. It suggests that the microenvironment in the area of damaged tissues is not conducive to support stem cell viability. One of the microenvironment is the hypoxia condition. Several scientific journals revealed that the administration of hypoxic cell culture can result in stress cells but on the other hand the stress condition of the cells also stimulates heat shock protein 27 (HSP 27 as antiapoptosis through inhibition of caspase 9. Purpose: The purpose of this study was to examine the role of heat shock protein 27 as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture. Methods: Stem cell culture was performed in hypoxic conditions (O2 1% and measured the resistance to apoptosis through HSP 27 and caspase 9 expression of bone marrow mesenchymal stem cells by using immunoflorecence and real time PCR. Results: The result of study showed that preconditioning hypoxia could inhibit apoptosis through increasing HSP 27 and decreasing level of caspase 9. Conclusion: The study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing caspase 9.Latar belakang: Konsep terapi stem cell merupakan salah satu harapan baru sebagai terapi medis kelainan kelenjar ludah. Namun, rendahnya viabilitas stem cell yang ditransplantasikan menyebabkan penurunan efektivitas terapi. Asumsi yang mendasari rendahnya viabilitas dan fungsi stem cell adalah tingginya kejadian apoptosis. Hal ini menunjukkan bahwa lingkungan mikro di daerah jaringan yang rusak tidak kondusif untuk mendukung viabilitas stem cell. Salah satu lingkungan

  7. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

    International Nuclear Information System (INIS)

    Reddy, A.S.N.

    2008-01-01

    Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of

  8. An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

    Science.gov (United States)

    Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

    2015-03-01

    As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  9. Impact of Heat Shock Protein A 12B Overexpression on Spinal Astrocyte Survival Against Oxygen-Glucose-Serum Deprivation/Restoration in Primary Cultured Astrocytes.

    Science.gov (United States)

    Xia, Xun; Ma, Yuan; Yang, Li-Bin; Cheng, Jing-Ming; Yang, Tao; Fan, Ke-Xia; Li, Yun-Ming; Liu, En-Yu; Cheng, Lin; Huang, Hai-Dong; Gu, Jian-Wen; Kuang, Yong-Qin

    2016-08-01

    Heat shock protein A 12B (HSPA12B) is a newly discovered member of the heat shock protein 70 family. Preclinical evidence indicates that HSPA12B helps protect the brain from ischemic injury, although its specific function remains unclear. The aim of this study is to investigate whether HSPA12B overexpression can protect astrocytes from oxygen-glucose-serum deprivation/restoration (OGD/R) injury. We analyzed the effects of HSPA12B overexpression on spinal cord ischemia-reperfusion injury and spinal astrocyte survival. After ischemia-reperfusion injury, we found that HSPA12B overexpression decreased spinal cord water content and infarct volume. MTT assay showed that HSPA12B overexpression increased astrocyte survival after OGD/R treatment. Flow cytometry results showed a marked inhibition of OGD/R-induced astrocyte apoptosis. Western blot assay showed that HSPA12B overexpression significantly increased regulatory protein B-cell lymphocyte 2 (Bcl-2) levels, whereas it decreased expression of the Bax protein, which forms a heterodimer with Bcl-2. Measurements of the level of activation of caspase-3 by Caspase-Glo®3/7 Assay kit showed that HSPA12B overexpression markedly inhibited caspase-3 activation. Notably, we demonstrated that the effects of HSPA12B on spinal astrocyte survival depended on activation of the PI3K/Akt signal pathway. These findings indicate that HSPA12B protects against spinal cord ischemia-reperfusion injury and may represent a potential treatment target.

  10. 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

  11. Heat Shock Proteins: Pathogenic Role in Atherosclerosis and Potential Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    Arman Kilic

    2012-01-01

    Full Text Available Heat shock proteins (HSPs are a highly conserved group of proteins that are constitutively expressed and function as molecular chaperones, aiding in protein folding and preventing the accumulation of misfolded proteins. In the arterial wall, HSPs have a protective role under normal physiologic conditions. In disease states, however, HSPs expressed on the vascular endothelial cell surface can act as targets for detrimental autoimmunity due to their highly conserved sequences. Developing therapeutic strategies for atherosclerosis based on HSPs is challenged by the need to balance such physiologic and pathologic roles of these proteins. This paper summarizes the role of HSPs in normal vascular wall processes as well as in the development and progression of atherosclerosis. The potential implications of HSPs in clinical therapies for atherosclerosis are also discussed.

  12. Overexpression of heat shock GroEL stress protein in leptospiral biofilm.

    Science.gov (United States)

    Vinod Kumar, K; Lall, Chandan; Vimal Raj, R; Vedhagiri, K; Kartick, C; Surya, P; Natarajaseenivasan, K; Vijayachari, P

    2017-01-01

    Leptospira is the causative agent of leptospirosis, which is an emerging zoonotic disease. Recent studies on Leptospira have demonstrated biofilm formation on abiotic surfaces. The protein expressed in the biofilm was investigated by using SDS-PAGE and immunoblotting in combination with MALDI-TOF mass spectrometry. The proteins expressed in Leptospira biofilm and planktonic cells was analyzed and compared. Among these proteins, one (60 kDa) was found to overexpress in biofilm as compared to the planktonic cells. MALDI-TOF analysis identified this protein as stress and heat shock chaperone GroEL. Our findings demonstrate that GroEL is associated with Leptospira biofilm. GroEL is conserved, highly immunogenic and a prominent stress response protein in pathogenic Leptospira spp., which may have clinical relevance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    Science.gov (United States)

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  14. 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

  15. Essential amino acid enriched high-protein enteral nutrition modulates insulin-like growth factor-1 system function in a rat model of trauma-hemorrhagic shock.

    Directory of Open Access Journals (Sweden)

    Xianfeng Xia

    Full Text Available Nutrition support for critically ill patients supplemented with additional modular protein may promote skeletal muscle protein anabolism in addition to counteracting acute nitrogen loss. The present study was designed to investigate whether the essential amino acid (EAA enriched high-protein enteral nutrition (EN modulates the insulin-like growth factor-1 (IGF-1 system and activates the mammalian target of rapamycin (mTOR anabolic signaling pathway in a trauma-hemorrhagic shock (T-HS rat model.Male Sprague-Dawley rats (n = 90, 278.18 ± 0.94 g were randomly assigned to 5 groups: (1 normal control, (2 pair-fed, (3 T-HS, (4 T-HS and standard EN, and (5 T-HS and EAA enriched high-protein EN. Six animals from each group were harvested on days 2, 4, and 6 for serum, gastrocnemius, soleus, and extensor digitorum longus sample collection. T-HS significantly reduced muscle mass. Nutrition support maintained muscle mass, especially the EAA enriched high-protein EN. Meanwhile, a pronounced derangement in IGF-1-IGFBPs axis as well as impaired mTOR transduction was observed in the T-HS group. Compared with animals receiving standard EN, those receiving EAA enriched high-protein EN presented 18% higher serum free IGF-1 levels following 3 days of nutrition support and 22% higher after 5 days. These changes were consistent with the concomitant elevation in serum insulin and reduction in corticosterone levels. In addition, phosphorylations of downstream anabolic signaling effectors - including protein kinase B, mTOR, and ribosomal protein S6 kinase1 - increased significantly in rats receiving EAA enriched high-protein EN.Our findings firstly demonstrate the beneficial effect of EAA enriched high-protein EN on the metabolic modulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction.

  16. Crystal structure of a small heat-shock protein from Xylella fastidiosa reveals a distinct high-order structure.

    Science.gov (United States)

    Fonseca, Emanuella Maria Barreto; Scorsato, Valéria; Dos Santos, Marcelo Leite; Júnior, Atilio Tomazini; Tada, Susely Ferraz Siqueira; Dos Santos, Clelton Aparecido; de Toledo, Marcelo Augusto Szymanski; de Souza, Anete Pereira; Polikarpov, Igor; Aparicio, Ricardo

    2017-04-01

    Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.

  17. Placental heat shock proteins: no immunohistochemical evidence for a differential stress response in preterm labour.

    Science.gov (United States)

    Divers, M J; Bulmer, J N; Miller, D; Lilford, R J

    1995-01-01

    The aetiology of idiopathic preterm labour remains obscure. The hypothesis that a stress response induced by low-grade bacterial infection in utero-placental tissues was investigated. Distribution of cognate and inducible isoforms of heat shock proteins (HSP) 70 kD, HSP 60 kD and HSP 90 kD were investigated in an immunohistochemical study of placental and decidual tissues before and after labour at varying gestations. Subjects were pregnant women undergoing singleton delivery after idiopathic preterm labour at less than 34 weeks' gestation (n = 23); spontaneous term labour at 37-42 weeks' gestation (n =24); preterm caesarean sections at less than 34 weeks' gestation for preeclampsia or intrauterine growth retardation (n=14); elective caesarean section at 37-42 weeks' gestation for cephalopelvic disproportion (n = 6). HSP expression was constant throughout the third trimester of pregnancy and did not change following the onset of labour, regardless of gestational age. A stress response in decidual tissues as determined by immunohistochemical analysis is apparently not associated with preterm labour.

  18. Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities

    Czech Academy of Sciences Publication Activity Database

    Durech, M.; Trčka, F.; Man, Petr; Blackburn, E.A.; Hernychová, L.; Dvořáková, P.; Coufalová, D.; Kavan, Daniel; Vojtěšek, B.; Muller, P.

    2016-01-01

    Roč. 15, č. 5 (2016), s. 1710-1727 ISSN 1535-9476 R&D Projects: GA MŠk(CZ) LO1509 EU Projects: Wellcome Trust(CZ) 01527/Z/13/Z Institutional support: RVO:61388971 Keywords : HEAT-SHOCK-PROTEIN * MOLECULAR CHAPERONE DNAK * SUBSTRATE-BINDING DOMAIN * INVASIVE BREAST - CANCER Subject RIV: CE - Biochemistry Impact factor: 6.540, year: 2016

  19. 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 ...

  20. Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12.

    Science.gov (United States)

    Trent, J D; Osipiuk, J; Pinkau, T

    1990-03-01

    The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins. In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, to a much lesser extent, two minor proteins (28 and 35 kilodaltons). Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant. They could not yet be related to known heat shock proteins. In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes. However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.

  1. 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...

  2. Heat shock proteins in relation to heat stress tolerance of creeping bentgrass at different N levels.

    Science.gov (United States)

    Wang, Kehua; Zhang, Xunzhong; Goatley, Mike; Ervin, Erik

    2014-01-01

    Heat stress is a primary factor causing summer bentgrass decline. Changes in gene expression at the transcriptional and/or translational level are thought to be a fundamental mechanism in plant response to environmental stresses. Heat stress redirects protein synthesis in higher plants and results in stress protein synthesis, particularly heat shock proteins (HSPs). The goal of this work was to analyze the expression pattern of major HSPs in creeping bentgrass (Agrostis stolonifera L.) during different heat stress periods and to study the influence of nitrogen (N) on the HSP expression patterns. A growth chamber study on 'Penn-A4' creeping bentgrass subjected to 38/28°C day/night for 50 days, was conducted with four nitrate rates (no N-0, low N-2.5, medium N-7.5, and high N-12.5 kg N ha-1) applied biweekly. Visual turfgrass quality (TQ), normalized difference vegetation index (NDVI), photochemical efficiency of photosystem II (Fv/Fm), shoot electrolyte leakage (ShEL), and root viability (RV) were monitored, along with the expression pattern of HSPs. There was no difference in measured parameters between treatments until week seven, except TQ at week five. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower ShEL and higher RV, suggesting a major role in improved heat tolerance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and their roles in bentgrass thermotolerance. In addition, their production seems to be resource dependent. This study could further improve our understanding about how different N levels affect bentgrass thermotolerance.

  3. Elevated expression of proto-oncogenes accompany enhanced induction of heat-shock genes after exposure of rat embryos in utero to ionizing irradiation

    International Nuclear Information System (INIS)

    Higo, H.; Lee, J.Y.; Satow, Y.; Higo, K.

    1989-01-01

    We have recently found that the effects of exposing rat embryos in utero to teratogens capable of producing cardiac anomalies were expressed later as enhanced induction of heat-shock proteins (hsp70 family) when embryonic hearts were cultured in vitro. However, it remained to be determined whether heat-shock proteins are induced in vivo after exposure to teratogens. The heat-shock response in some mammalian systems is known to be accompanied by elevated expression of proto-oncogenes. Using gene-specific DNA probes, we examined the levels of the expression (transcription) of heat-shock protein genes and two nuclear proto-oncogenes, c-fos and c-myc, in the embryos removed from irradiated pregnant mother rats 4 or 5 days after the irradiation. We found that the levels of expression in vivo of the hsp70 and c-myc genes in the irradiated embryos increased by approximately twofold as compared with those in the control. The expression in vivo of the c-fos gene was not detected in either the irradiated or non-irradiated embryos. After 0.5-hr incubation in vitro of the embryos, however, the expression of the c-fos gene in the irradiated embryos was highly enhanced whereas the control showed no changes. Although the exact functions of these gene products still remain obscure, the enhanced expression of hsp70 gene(s) and the nuclear proto-oncogenes observed in the present study may reflect repair of intracellular damages and/or regeneration of tissue by compensatory cell proliferation, processes that may disturb the normal program of organogenesis

  4. Shock disturbance of the I-Xe system

    International Nuclear Information System (INIS)

    Caffee, M.W.; Hohenberg, C.M.; Podosek, F.A.; Swindle, T.D.

    1982-01-01

    Three separate samples of the meteorite Bjurbole were artificially shocked at pressures of 70 kb, 200 kb, and 400 kb. Analysis of xenon released in stepwise heating shows that the I-Xe system of the 400 kb sample is substantially altered by the shock loading, and it is no longer possible to infer an age or trapped xenon composition for that sample. The 200-kb and 70-kb samples display isotopic structures progressively less altered demonstrating the gradations in shock disturbance likely to be found in natural systems. Interpretations of the I-Xe and Ar-40-Ar-39 systems for several naturally shocked meteorites are also presented. New data for Arapahoe do not confirm the previously reported age and trapped xenon composition, demonstrating instead that its I-Xe structure has been strongly disturbed by shock

  5. Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the α-crystallin family

    Energy Technology Data Exchange (ETDEWEB)

    Hilario, Eduardo; Teixeira, Elaine Cristina; Pedroso, Gisele Audrei; Bertolini, Maria Célia [Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil); Medrano, Francisco Javier, E-mail: fjmedrano@yahoo.com [Departamento de Cristalografia de Proteínas, Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13084-971, Campinas-SP (Brazil); Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil)

    2006-05-01

    XAC1151, a small heat-shock protein from X. axonopodis pv. citri belonging to the α-crystallin family, was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 Å. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

  6. Effects of exposing rat embryos in utero to physical or chemical teratogens are expressed later as enhanced induction of heat-shock proteins when embryonic hearts are cultured in vitro

    International Nuclear Information System (INIS)

    Higo, H.; Higo, K.; Lee, J.Y.; Hori, H.; Satow, Y.

    1988-01-01

    In order to get more insight into the effects of teratogens on developing embryos, we investigated the protein synthesis patterns of the target organs isolated from teratogen-treated embryos. Rat embryos were either irradiated in utero with either 252Cf fission neutrons or 60Co gamma rays on day 8 of gestation or treated in utero with a bis(dichloroacetyl)diamine (a chemical teratogen) on days 9 and 10. Hearts were removed from the embryos on day 12 and were incubated in vitro at 37 degrees C in the presence of [35S]methionine for up to 8 hr. The newly synthesized labeled proteins were then analyzed qualitatively by two-dimensional polyacrylamide gel electrophoresis. Enhanced and prolonged induction of a family of heat-shock (stress) proteins with a molecular weight of about 70,000 (SP70s) was observed as compared with those of controls. Among the teratogen-treated hearts, those with gross malformations already detectable at this early stage showed especially higher inductions of SP70s than did the others. The abnormal expression of SP70s observed in the present study appears to be a reflection of persisting cellular (tissue) damage inflicted by the teratogens, and the extent of the induction may be indicative of the degree and/or type of the damage. Such persisting defects in surviving cells, manifested by abnormal induction of SP70s in the present study, might be related to malformation of embryonic hearts

  7. Errors in macromolecular synthesis after stress. A study of the possible protective role of the small heat shock proteinsBiochemistry

    NARCIS (Netherlands)

    Marin Vinader, L.

    2006-01-01

    The general goal of this thesis was to gain insight in what small heat shock proteins (sHsps) do with respect to macromolecular synthesis during a stressful situation in the cell. It is known that after a non-lethal heat shock, cells are better protected against a subsequent more severe heat shock,

  8. Prediction of massive bleeding. Shock index and modified shock index.

    Science.gov (United States)

    Terceros-Almanza, L J; García-Fuentes, C; Bermejo-Aznárez, S; Prieto-Del Portillo, I J; Mudarra-Reche, C; Sáez-de la Fuente, I; Chico-Fernández, M

    2017-12-01

    To determine the predictive value of the Shock Index and Modified Shock Index in patients with massive bleeding due to severe trauma. Retrospective cohort. Severe trauma patient's initial attention at the intensive care unit of a tertiary hospital. Patients older than 14 years that were admitted to the hospital with severe trauma (Injury Severity Score >15) form January 2014 to December 2015. We studied the sensitivity (Se), specificity (Sp), positive and negative predictive value (PV+ and PV-), positive and negative likelihood ratio (LR+ and LR-), ROC curves (Receiver Operating Characteristics) and the area under the same (AUROC) for prediction of massive hemorrhage. 287 patients were included, 76.31% (219) were male, mean age was 43,36 (±17.71) years and ISS was 26 (interquartile range [IQR]: 21-34). The overall frequency of massive bleeding was 8.71% (25). For Shock Index: AUROC was 0.89 (95% confidence intervals [CI] 0.84 to 0.94), with an optimal cutoff at 1.11, Se was 91.3% (95% CI: 73.2 to 97.58) and Sp was 79.69% (95% CI: 74.34 to 84.16). For the Modified Shock Index: AUROC was 0.90 (95% CI: 0.86 to 0.95), with an optimal cutoff at 1.46, Se was 95.65% (95% CI: 79.01 to 99.23) and Sp was 75.78% (95% CI: 70.18 to 80.62). Shock Index and Modified Shock Index are good predictors of massive bleeding and could be easily incorporated to the initial workup of patients with severe trauma. Copyright © 2017 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.

  9. Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

    Science.gov (United States)

    Endo, H; Yano, M; Okumura, Y; Kido, H

    2014-01-01

    Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance. PMID:24481441

  10. Heat shock protein 10 (Hsp10) in immune-related diseases: one coin, two sides

    Science.gov (United States)

    Jia, Haibo; Halilou, Amadou I.; Hu, Liang; Cai, Wenqian; Liu, Jing; Huang, Bo

    2011-01-01

    Heat shock protein 10 (Hsp10) in eukaryotes, originally identified as a mitochondrial chaperone, now is also known to be present in cytosol, cell surface, extracellular space and peripheral blood. Functionally besides participating in mitochondrial protein folding in association with Hsp60, Hsp10 appears to be related to pregnancy, cancer and autoimmune inhibition. Hsp10 can be released to peripheral blood at very early time point of pregnancy and given another name called early pregnancy factor (EPF), which seems to play a critical role in developing a pregnant niche. In malignant disorders, Hsp10 is usually abnormally expressed in the cytosol of malignant cells and further released to extracellular space, resulting in tumor-promoting effect from various aspects. Furthermore, distinct from other heat shock protein members, whose soluble form is recognized as danger signal by immune cells and triggers immune responses, Hsp10 after release, however, is designed to be an inhibitory signal by limiting immune response. This review discusses how Hsp10 participates in various physiological and pathological processes from basic protein molecule folding to pregnancy, cancer and autoimmune diseases, and emphasizes how important the location is for the function exertion of a molecule. PMID:21969171

  11. Linking physiological and cellular responses to thermal stress: β-adrenergic blockade reduces the heat shock response in fish.

    Science.gov (United States)

    Templeman, Nicole M; LeBlanc, Sacha; Perry, Steve F; Currie, Suzanne

    2014-08-01

    When faced with stress, animals use physiological and cellular strategies to preserve homeostasis. We were interested in how these high-level stress responses are integrated at the level of the whole animal. Here, we investigated the capacity of the physiological stress response, and specifically the β-adrenergic response, to affect the induction of the cellular heat shock proteins, HSPs, following a thermal stress in vivo. We predicted that blocking β-adrenergic stimulation during an acute heat stress in the whole animal would result in reduced levels of HSPs in red blood cells (RBCs) of rainbow trout compared to animals where adrenergic signaling remained intact. We first determined that a 1 h heat shock at 25 °C in trout acclimated to 13 °C resulted in RBC adrenergic stimulation as determined by a significant increase in cell swelling, a hallmark of the β-adrenergic response. A whole animal injection with the β2-adrenergic antagonist, ICI-118,551, successfully reduced this heat-induced RBC swelling. The acute heat shock caused a significant induction of HSP70 in RBCs of 13 °C-acclimated trout as well as a significant increase in plasma catecholamines. When heat-shocked fish were treated with ICI-118,551, we observed a significant attenuation of the HSP70 response. We conclude that circulating catecholamines influence the cellular heat shock response in rainbow trout RBCs, demonstrating physiological/hormonal control of the cellular stress response.

  12. Aggregation of SND1 in Stress Granules is Associated with the Microtubule Cytoskeleton During Heat Shock Stimulus.

    Science.gov (United States)

    Shao, Jie; Gao, Fei; Zhang, Bingbing; Zhao, Meng; Zhou, Yunli; He, Jinyan; Ren, Li; Yao, Zhi; Yang, Jie; Su, Chao; Gao, Xingjie

    2017-12-01

    Stress granules (SGs) are dynamic dense structures in the cytoplasm that form in response to a variety of environmental stress stimuli. Staphylococcal nuclease and Tudor domain containing 1 (SND1) is a type of RNA-binding protein and has been identified as a transcriptional co-activator. Our previous studies have shown that SND1 is a component of the stress granule, which forms under stress conditions. Here, we observed that SND1 granules were often surrounded by ɑ-tubulin-microtubules in 45°C-treated HeLa cells at 15 min or colocalized with microtubules at 30 or 45 min. Furthermore, Nocodazole-mediated microtubule depolymerization could significantly affect the efficient recruitment of SND1 proteins to the SGs during heat shock stress. In addition, the 45°C heat shock mediated the enhancement of eIF2α phosphorylation, which was not affected by treatment with Nocodazole, an agent that disrupts the cytoskeleton. The intact microtubule cytoskeletal tracks are important for the efficient assembly of SND1 granules under heat shock stress and may facilitate SND1 shuttling between cytoplasmic RNA foci. Anat Rec, 300:2192-2199, 2017. © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists. Copyright © 2017 The Authors The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

  13. Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yun-Jeong; Cho, Hyun-Ji [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Magae, Junji [Magae Bioscience Institute, 49-4 Fujimidai, Tsukuba 300-1263 (Japan); Lee, In-Kyu [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Park, Keun-Gyu, E-mail: kpark@knu.ac.kr [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2013-12-15

    Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.

  14. 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.

  15. Small heat shock protein αA-crystallin prevents photoreceptor degeneration in experimental autoimmune uveitis.

    Directory of Open Access Journals (Sweden)

    Narsing A Rao

    Full Text Available The small heat shock protein, αA-crystallin null (αA-/- mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU. In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB, a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA-/- and wild type mice with EAU as donors and Rag2-/- as the recipient mice, which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ, both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses.

  16. Small heat shock protein αA-crystallin prevents photoreceptor degeneration in experimental autoimmune uveitis.

    Science.gov (United States)

    Rao, Narsing A; Saraswathy, Sindhu; Pararajasegaram, Geeta; Bhat, Suraj P

    2012-01-01

    The small heat shock protein, αA-crystallin null (αA-/-) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA-/- and wild type mice with EAU as donors and Rag2-/- as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses.

  17. 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)

  18. The induction of heme oxygenase-1 suppresses heat shock protein 90 and the proliferation of human breast cancer cells through its byproduct carbon monoxide

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Wen-Ying [Department of Pathology, Chi-Mei Hospital, Tainan, Taiwan (China); Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Chen, Yen-Chou [Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Shih, Chwen-Ming; Lin, Chun-Mao; Cheng, Chia-Hsiung; Chen, Ku-Chung [Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Biochemistry, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Cheng-Wei, E-mail: cwlin@tmu.edu.tw [Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Biochemistry, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

    2014-01-01

    Heme oxygenase (HO)-1 is an oxidative stress-response enzyme which catalyzes the degradation of heme into bilirubin, ferric ion, and carbon monoxide (CO). Induction of HO-1 was reported to have antitumor activity; the inhibitory mechanism, however, is still unclear. In the present study, we found that treatment with [Ru(CO){sub 3}Cl{sub 2}]{sub 2} (RuCO), a CO-releasing compound, reduced the growth of human MCF7 and MDA-MB-231 breast cancer cells. Analysis of growth-related proteins showed that treatment with RuCO down-regulated cyclinD1, CDK4, and hTERT protein expressions. Interestingly, RuCO treatment resulted in opposite effects on wild-type and mutant p53 proteins. These results were similar to those of cells treated with geldanamycin (a heat shock protein (HSP)90 inhibitor), suggesting that RuCO might affect HSP90 activity. Moreover, RuCO induced mutant p53 protein destabilization accompanied by promotion of ubiquitination and proteasome degradation. The induction of HO-1 by cobalt protoporphyrin IX (CoPP) showed consistent results, while the addition of tin protoporphyrin IX (SnPP), an HO-1 enzymatic inhibitor, diminished the RuCO-mediated effect. RuCO induction of HO-1 expression was reduced by a p38 mitogen-activated protein kinase inhibitor (SB203580). Additionally, treatment with a chemopreventive compound, curcumin, induced HO-1 expression accompanied with reduction of HSP90 client protein expression. The induction of HO-1 by curcumin inhibited 12-O-tetradecanoyl-13-acetate (TPA)-elicited matrix metalloproteinase-9 expression and tumor invasion. In conclusion, we provide novel evidence underlying HO-1's antitumor mechanism. CO, a byproduct of HO-1, suppresses HSP90 protein activity, and the induction of HO-1 may possess potential as a cancer therapeutic. - Highlights: • CO and HO-1 inhibited the growth of human breast cancer cells. • CO and HO-1 attenuated HSP90 and its client proteins expression. • CO induced mutant p53 protein

  19. Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90

    Directory of Open Access Journals (Sweden)

    Alex Henke

    2016-08-01

    Full Text Available Background: Prostate cancer-associated fibroblasts (CAF can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix; Methods: We isolated CAF from prostate cancer patients of Gleason Score 6–10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90 inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay; Results: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFβ2 levels in CAF; Conclusions: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.

  20. Prognostic implications of carboxyl-terminus of Hsc70 interacting protein and lysyl-oxidase expression in human breast cancer

    Directory of Open Access Journals (Sweden)

    Patani Neill

    2010-01-01

    Full Text Available Background: Ubiquitin modification of proteins influences cellular processes relevant to carcinogenesis. CHIP (carboxyl-terminus of Hsc70-interacting protein is a chaperone-dependent E3 ubiquitin ligase, regulating the stability of heat shock protein 90 (HSP90 interacting proteins. CHIP is implicated in the modulation of estrogen receptor (ESR1 and Her-2/neu (ERBB2 stability. LOX (lysyl-oxidase serves intracellular roles and catalyses the cross-linking of extracellular matrix (ECM collagens and elastin. LOX expression is altered in human malignancies and their peri-tumoral stroma. However, paradoxical roles are reported. In this study, the level of mRNA expression of CHIP and LOX were assessed in normal and malignant breast tissue and correlated with clinico-pathological parameters. Materials and Methods: Breast cancer (BC tissues (n = 127 and normal tissues (n = 33 underwent RNA extraction and reverse transcription; transcript levels were determined using real-time quantitative PCR and normalized against CK-19. Transcript levels were analyzed against TNM stage, nodal involvement, tumor grade and clinical outcome over a ten-year follow-up period. Results: CHIP expression decreased with increasing Nottingham Prognostic Index (NPI: NPI-1 vs. NPI-3 (12.2 vs. 0.2, P = 0.0264, NPI-2 vs. NPI-3 (3 vs. 0.2, P = 0.0275. CHIP expression decreased with increasing TNM stage: TNM-1 vs. TNM-2 (12 vs. 0, P = 0.0639, TNM-1 vs. TNM-2-4 (12 vs. 0, P = 0.0434. Lower transcript levels were associated with increasing tumor grade: grade 1 vs. grade 3 (17.7 vs. 0.3, P = 0.0266, grade 2 vs. grade 3 (5 vs. 0.3, P = 0.0454. The overall survival (OS for tumors classified as ′low-level expression′, was poorer than those with ′high-level expression′ (118.1 vs. 152.3 months, P = 0.039. LOX expression decreased with increasing NPI: NPI-1 vs. NPI-2 (3 vs. 0, P = 0.0301 and TNM stage: TNM-1 = 3854639, TNM-2 = 908900, TNM-3 = 329, TNM-4 = 1.232 (P = NS. Conclusion: CHIP

  1. 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

  2. Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

    Science.gov (United States)

    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.

  3. 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.

  4. Synthesis of 19-substituted geldanamycins with altered conformations and their binding to heat shock protein Hsp90

    Science.gov (United States)

    Kitson, Russell R. A.; Chang, Chuan-Hsin; Xiong, Rui; Williams, Huw E. L.; Davis, Adrienne L.; Lewis, William; Dehn, Donna L.; Siegel, David; Roe, S. Mark; Prodromou, Chrisostomos; Ross, David; Moody, Christopher J.

    2013-01-01

    The benzoquinone ansamycin geldanamycin and its derivatives are inhibitors of heat shock protein Hsp90, an emerging target for novel therapeutic agents both in cancer and in neurodegeneration. However, toxicity of these compounds to normal cells has been ascribed to reaction with thiol nucleophiles at the quinone 19-position. We reasoned that blocking this position would ameliorate toxicity, and that it might also enforce a favourable conformational switch of the trans-amide group into the cis-form required for protein binding. We report here an efficient synthesis of such 19-substituted compounds and realization of our hypotheses. Protein crystallography established that the new compounds bind to Hsp90 with, as expected, a cis-amide conformation. Studies on Hsp90 inhibition in cells demonstrated the molecular signature of Hsp90 inhibitors: decreases in client proteins with compensatory increases in other heat shock proteins in both human breast cancer and dopaminergic neural cells, demonstrating their potential for use in the therapy of cancer or neurodegenerative diseases. PMID:23511419

  5. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-05-01

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  6. Effects of thermal stress of protein synthesis and gene expression in Brassica napus

    International Nuclear Information System (INIS)

    Halle, J.R.; Ghosh, S.; Dumbroff, E.B.; Heikkila, J.J.

    1989-01-01

    Leaf segments of Brassica napus were exposed to 22 degrees, 35 degrees, 38 degrees or 40 degrees C for up to 4 h. Analysis of radiolabelled proteins by 2-D SDS-PAGE and fluorography revealed two major groups of heat shock proteins (HSPs). One group comprised HSPs, 70, 76 and 87, with pIs ranging from 5.7 to 6.1, whereas the second group had molecular weights ranging from 23 to 16 kD and pIs from 5.6 to 6.9. Immunoblot analysis using antibodies directed against the large (RLSU) and small (RSSU) subunits of ribulose-1,5-bisphosphate carboxylase (RUBISCO) showed that increasing temperatures from 35 degrees to 38 degrees or 40 degrees C or the duration of thermal stress from 1 to 5 h did not affect levels of the RSSU (15 kd) whereas levels of the RLSU (52 kD) fell sharply. Nevertheless, RUBISCO activity was not adversely affected at 38 degree C for periods of up to 5 h. The increase observed in HSP 70 during heat shock was transcriptionally regulated, but the decrease in the RLSU was not accompanied by any detectable change in levels of its mRNA

  7. Heat shock protein 70 gene polymorphisms are associated with paranoid schizophrenia in the Polish population.

    Science.gov (United States)

    Kowalczyk, Malgorzata; Owczarek, Aleksander; Suchanek, Renata; Paul-Samojedny, Monika; Fila-Danilow, Anna; Borkowska, Paulina; Kucia, Krzysztof; Kowalski, Jan

    2014-03-01

    HSP70 genes have been considered as promising schizophrenia candidate genes based on their protective role in the central nervous system under stress conditions. In this study, we analyzed the potential implication of HSPA1A +190G/C, HSPA1B +1267A/G, and HSPA1L +2437T/C polymorphisms in the susceptibility to paranoid schizophrenia in a homogenous Caucasian Polish population. In addition, we investigated the association of the polymorphisms with the clinical variables of the disease. Two hundred and three patients with paranoid schizophrenia and 243 healthy controls were enrolled in the study. Polymorphisms of HSPA1A, -1B, and -1L genes were genotyped using the PCR-RFLP technique. Analyses were conducted in entire groups and in subgroups that were stratified according to gender. There were significant differences in the genotype and allele frequencies of HSPA1A polymorphism between the patients and controls. The +190CC genotype and +190C allele were over-represented in the patients and significantly increased the risk for developing schizophrenia (OR = 3.45 and OR = 1.61, respectively). Interestingly, such a risk was higher for females with the +190CC genotype than for males with the +190CC genotype (OR = 5.78 vs. OR = 2.76). We also identified the CGT haplotype as a risk haplotype for schizophrenia and demonstrated the effects of HSPA1A and HSPA1B genotypes on the psychopathology and age of onset. Our study provided the first evidence that the HSPA1A polymorphism may potentially increase the risk of developing paranoid schizophrenia. Further independent analyses in different populations to evaluate the role of gender are needed to replicate these results.

  8. 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

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    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.

  9. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

  10. Ribulose 1,5-bisphosphate carboxylase synthesis during heat shock

    International Nuclear Information System (INIS)

    Vierling, E.; Key, J.L.

    1985-01-01

    Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 0 C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 0 C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 0 C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. The authors conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.,S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mNRA levels under the conditions examined

  11. The small heat shock proteins from Acidithiobacillus ferrooxidans: gene expression, phylogenetic analysis, and structural modeling

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    Ribeiro Daniela A

    2011-12-01

    Full Text Available Abstract Background Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms. Results The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell. Conclusion We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.

  12. Purification of cold-shock-like proteins from Stigmatella aurantiaca - molecular cloning and characterization of the cspA gene.

    Science.gov (United States)

    Stamm, I; Leclerque, A; Plaga, W

    1999-09-01

    Prominent low-molecular-weight proteins were isolated from vegetative cells of the myxobacterium Stigmatella aurantiaca and were found to be members of the cold-shock protein family. A first gene of this family (cspA) was cloned and sequenced. It encodes a protein of 68 amino acid residues that displays up to 71% sequence identity with other bacterial cold-shock(-like) proteins. A cysteine residue within the RNP-2 motif is a peculiarity of Stigmatella CspA. A cspA::(Deltatrp-lacZ) fusion gene construct was introduced into Stigmatella by electroporation, a method that has not been used previously for this strain. Analysis of the resultant transformants revealed that cspA transcription occurs at high levels during vegetative growth at 20 and 32 degrees C, and during fruiting body formation.

  13. Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas

    International Nuclear Information System (INIS)

    Warchalowska-Sliwa, E.; Niklinska, M.; Goerlich, A.; Michailova, P.; Pyza, E.

    2005-01-01

    The orthopteran insect Tetrix tenuicornis, collected from polluted and unpolluted areas, was used to study heavy metal accumulation and its impact on stress protein levels and on changes in the number and morphology of chromosomes in mitotic and meiotic cells. During two consecutive years, insects were collected from polluted areas of zinc-lead mine spoils near Boleslaw (Poland) and from unpolluted areas near Busko and Staszow (Poland). T. tenuicornis from the polluted area showed 1.5, 4.03, 4.32 and 41.73 times higher concentrations of copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd), respectively, than insects of the same species collected from unpolluted areas. Insects exposed to heavy metals showed only small changes, and rather a decrease in the concentration of constitutive and inducible heat shock proteins Hsp70, the level of which increases under stress conditions. A cytogenetic study of T. tenuicornis revealed intra-population anomalies in chromosome number and morphology in mitotic and meiotic cells and the presence of an additional B chromosome in germinal cells. In 50% of females collected from polluted areas, mosaic oogonial mitotic chromosome sets and diploid, hypo- or hypertetraploid, tetraploid, and octoploid chromosome numbers were detected. In turn, 14.6% of males showed a heterozygous deficiency of chromatin in L 2 and M 3 bivalents in addition to the presence of B chromosomes. - Metals accumulation caused genotoxicity in insects

  14. 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.

  15. Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

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    Alessia Arcaro

    2015-01-01

    Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

  16. 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.

  17. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

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    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  18. A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

    Science.gov (United States)

    Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

    2016-11-16

    Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  19. Heat shock protein 27 phosphorylation state is associated with cancer progression

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    Maria eKatsogiannou

    2014-10-01

    Full Text Available Understanding the mechanisms that control stress-induced survival is critical to explain how tumors frequently resist to treatment and to improve current anti-cancer therapies. Cancer cells are able to cope with stress and escape drug toxicity by regulating heat shock proteins (Hsps expression and function. Hsp27 (HSPB1, a member of the small Hsp family, represents one of the key players of many signaling pathways contributing to tumorigenicity, treatment resistance and apoptosis inhibition. Hsp27 is overexpressed in many types of cancer and its functions are regulated by post-translational modifications, such as phosphorylation. Protein phosphorylation is the most widespread signaling mechanism in eukaryotic cells, and it is involved in all fundamental cellular processes. Aberrant phosphorylation of Hsp27 has been associated with several diseases such as cancer but the molecular mechanisms by which it is implicated in cancer development and progression remain undefined. This review focuses on the role of phosphorylation in Hsp27 functions in cancer cells and its potential usefulness as therapeutic target in cancer.

  20. 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.