Sample records for citrus hsp70 molecular

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

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    Luciano G. Fietto


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

  2. The nucleotide exchange factors of Hsp70 molecular chaperone

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    Andreas eBracher


    Full Text Available Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1 and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell.

  3. The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex. (United States)

    Trcka, Filip; Durech, Michal; Man, Petr; Hernychova, Lenka; Muller, Petr; Vojtesek, Borivoj


    Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

  4. Molecular cloning and functional analysis of the drought tolerance gene MsHSP70 from alfalfa (Medicago sativa L.). (United States)

    Li, Zhenyi; Long, Ruicai; Zhang, Tiejun; Wang, Zhen; Zhang, Fan; Yang, Qingchuan; Kang, Junmei; Sun, Yan


    Heat shock proteins (HSPs) are a ubiquitously expressed class of protective proteins that play a key role in plant response to stressful conditions. This study aimed to characterize and investigate the function of an HSP gene in alfalfa (Medicago sativa). MsHSP70, which contains a 2028-bp open reading frame, was identified through homology cloning. MsHSP70 shares high sequence identity (94.47%) with HSP70 from Medicago truncatula. Expression analysis of MsHSP70 in alfalfa organs revealed a relatively higher expression level in aerial organs such as flowers, stems and leaves than in roots. MsHSP70 was induced by heat shock, abscisic acid (ABA) and hydrogen peroxide. Transgenic Arabidopsis seedlings overexpressing MsHSP70 were hyposensitive to polyethylene glycol (PEG) and ABA treatments, suggesting that exogenous expression of MsHSP70 enhanced Arabidopsis tolerance to these stresses. Examination of physiological indexes related to drought and ABA stress demonstrated that in comparison with non-transgenic plants, T3 transgenic Arabidopsis plants had an increased proline content, higher superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content. Furthermore, higher relative water content (RWC) was detected in transgenic plants compared with non-transgenic plants under drought stress. These findings clearly indicate that molecular manipulation of MsHSP70 in plants can have substantial effects on stress tolerance.

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

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    Pappas Georgios J


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

  6. The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease. (United States)

    Mohanan, Vishnu; Grimes, Catherine Leimkuhler


    Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.

  7. Thermal tolerance of the crab Pachygrapsus marmoratus: intraspecific differences at a physiological (CTMax) and molecular level (Hsp70). (United States)

    Madeira, D; Narciso, L; Cabral, H N; Diniz, M S; Vinagre, C


    Temperature is one of the most important variables influencing organisms, especially in the intertidal zone. This work aimed to test physiological and molecular intraspecific differences in thermal tolerance of the crab Pachygrapsus marmoratus (Fabricius, 1787). The comparisons made focused on sex, size, and habitat (estuary and coast) differences. The physiological parameter was upper thermal limit, tested via the critical thermal maximum (CTMax) and the molecular parameter was total heat shock protein 70 (Hsp70 and Hsp70 plus Hsc70) production, quantified via an enzyme-linked imunosorbent assay. Results showed that CTMax values and Hsp70 production are higher in females probably due to different microhabitat use and potentially due to different hormonal regulation in males and females. Among females, non-reproducing ones showed a higher CTMax value, but no differences were found in Hsp70, even though reproducing females showed higher variability in Hsp70 amounts. As reproduction takes up a lot of energy, its allocation for other activities, including stress responses, is lower. Juveniles also showed higher CTMax and Hsp70 expression because they occur in greater shore heights and ageing leads to alterations in protein synthesis. Comparing estuarine and coastal crabs, no differences were found in CTMax but coastal crabs produce more Hsp70 than estuarine crabs because they occur in drier and hotter areas than estuarine ones, which occur in moister environments. This work shows the importance of addressing intraspecific differences in the stress response at different organizational levels. This study shows that these differences are key factors in stress research, climate research, and environmental monitoring.

  8. Molecular cloning of HSP70 in Mycoplasma ovipneumoniae and comparison with that of other mycoplasmas. (United States)

    Li, M; Ma, C J; Liu, X M; Zhao, D; Xu, Q C; Wang, Y J


    Mycoplasma ovipneumoniae, a bacterial species that specifically affects ovine and goat, is the cause of ovine infectious pleuropneumonia. We cloned, sequenced and analyzed heat shock protein 70 (HSP70) (dnaK) gene of M. ovipneumoniae. The full length open reading frame of the M. ovipneumoniae HSP70 gene consists of 1812 nucleotides, with a G+C content of 34.16%, encoding 604 amino acids. Comparative analysis with the HSP70 sequences of 15 Mycoplasma species revealed 59 to 87% DNA sequence identity, with an amino acid sequence identity range of 58 to 94%. M. ovipneumoniae and M. hyopneumoniae shared the highest DNA and amino acid sequence identity (87 and 94%, respectively). Based on phylogenetic analysis, both the DNA and amino acid identities of M. ovipneumoniae with other mycoplasmal HSP70 were correlated with the degree of relationship between the species. The C-terminus of the HSP70 was cloned into a bacterial expression vector and expressed in Escherichia coli cells. The recombinant C-terminal portion of HSP70 protein strongly reacted with convalescent sera from M. ovipneumoniae-infected sheep, based on an immunoblotting assay. This indicates that HSP70 is immunogenic in a natural M. ovipneumoniae infection and may be a relevant antigen for vaccine development.

  9. Molecular characterization and expression of HSP70, HSF and HSBP genes inOctopus vulgaris during thermal stress

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    HONG Jingni; MAO Yong; NIU Sufang; SUN Tiantian; SU Yongquan


    Temperature is an important environmental factor that affects the growth and survival ofOctopus vulgaris, the common octopus. To understand the protective mechanism thatO.vulgaris exhibits under heat stress, we used rapid amplification of cDNA ends (RACE) to obtain full-length sequences of three heat stress response related genes: (1) the heat shock protein 70 (OvHSP70), (2) the heat shock transcription factor (OvHSF) , and (3) the heat shock factor-binding protein (OvHSBP) ofO.vulgaris. The OvHSP70, OvHSF, and OvHSBP proteins contained 2 222 bp, 2 264 bp, 841 bp that encoded for 635, 458 and 90 amino acids, respectively. The results of multiple sequence alignment showed that the amino acid sequences of OvHSP70 were highly conserved with respect to other species. Similarly, the DNA binding domain, the trimerization domain of OvHSF, and the coiled coil region of OvHSBP also had highly conserved regions. The real-time polymerase chain reaction (PCR) results indicated that OvHSP70 was temperature-dependent and time-dependent, showing a positive response to heat stress. On exposure to 28°C and to 30°C, the mRNA expression levels of OvHSF and OvHSBP were higher than those in the control group at 24°C. The mRNA expression of OvHSBP significantly increased with heat treatment at 26°C, while the mRNA expression of OvHSF decreased. The experimental results indicated that the expression of OvHSP70, OvHSF and OvHSBP were all sensitive to heat stress, which suggests that these three genes may play an important role forO.vulgaris in responding to environmental stress. Thus, this study sets a theoretical foundation for further in-depth studies on the molecular protective mechanisms of the heat response inO.vulgaris.

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

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    CB Toaldo


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

  11. Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones (United States)

    Finka, Andrija; Sharma, Sandeep K.; Goloubinoff, Pierre


    Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases. PMID:26097841

  12. The molecular chaperone Hsp70 activates protein phosphatase 5 (PP5) by binding the tetratricopeptide repeat (TPR) domain. (United States)

    Connarn, Jamie N; Assimon, Victoria A; Reed, Rebecca A; Tse, Eric; Southworth, Daniel R; Zuiderweg, Erik R P; Gestwicki, Jason E; Sun, Duxin


    Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.

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

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    Requena Jose M


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

  14. Functional analysis of Hsp70 inhibitors.

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    Rainer Schlecht

    Full Text Available The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES, which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1 and constitutive Hsc70 (HSPA8 is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD. Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.

  15. Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion. (United States)

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


    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.

  16. The Complex Function of Hsp70 in Metastatic Cancer

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    Kata Juhasz


    Full Text Available 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.

  17. The Complex Function of Hsp70 in Metastatic Cancer

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


    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.

  18. Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

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    Luong Michael


    Full Text Available Abstract Background Toll-like receptor 4 (TLR4 is activated by bacterial endotoxin, a prototypical pathogen-associated molecular pattern (PAMP. It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification. Results Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein as determined by ELISA. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls. Conclusions These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

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

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    Adélle Burger


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

  20. The conformational dynamics of the mitochondrial Hsp70 chaperone. (United States)

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


    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.

  1. Metazoan Hsp70-based protein disaggregases: Emergence and mechanisms

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    Nadinath Bandara Nillegoda


    Full Text Available Proteotoxic stresses and ageing cause breakdown of cellular protein homeostasis, allowing misfolded proteins to form aggregates, which dedicated molecular machines have evolved to solubilize. In bacteria, fungi, protozoa and plants protein disaggregation involves an Hsp70-J-protein chaperone system, which loads and activates a powerful AAA+ ATPase (Hsp100 disaggregase onto protein aggregate substrates. Metazoans lack cytosolic and nuclear Hsp100 disaggregases but still eliminate protein aggregates. This longstanding puzzle of protein quality control is now resolved. Robust protein disaggregation activity recently shown for the metazoan Hsp70-based disaggregases relies instead on a crucial cooperation between two J-protein classes and interaction with the Hsp110 co-chaperone. An expanding multiplicity of Hsp70 and J-protein family members in metazoan cells facilitates different configurations of this Hsp70-based disaggregase allowing unprecedented versatility and specificity in protein disaggregation. Here we review the architecture, operation and adaptability of the emerging metazoan disaggregation system and discuss how this evolved.

  2. The complex evolutionary dynamics of Hsp70s: a genomic and functional perspective. (United States)

    Kominek, Jacek; Marszalek, Jaroslaw; Neuvéglise, Cécile; Craig, Elizabeth A; Williams, Barry L


    Hsp70 molecular chaperones are ubiquitous. By preventing aggregation, promoting folding, and regulating degradation, Hsp70s are major factors in the ability of cells to maintain proteostasis. Despite a wealth of functional information, little is understood about the evolutionary dynamics of Hsp70s. We undertook an analysis of Hsp70s in the fungal clade Ascomycota. Using the well-characterized 14 Hsp70s of Saccharomyces cerevisiae, we identified 491 orthologs from 53 genomes. Saccharomyces cerevisiae Hsp70s fall into seven subfamilies: four canonical-type Hsp70 chaperones (SSA, SSB, KAR, and SSC) and three atypical Hsp70s (SSE, SSZ, and LHS) that play regulatory roles, modulating the activity of canonical Hsp70 partners. Each of the 53 surveyed genomes harbored at least one member of each subfamily, and thus establishing these seven Hsp70s as units of function and evolution. Genomes of some species contained only one member of each subfamily that is only seven Hsp70s. Overall, members of each subfamily formed a monophyletic group, suggesting that each diversified from their corresponding ancestral gene present in the common ancestor of all surveyed species. However, the pattern of evolution varied across subfamilies. At one extreme, members of the SSB subfamily evolved under concerted evolution. At the other extreme, SSA and SSC subfamilies exhibited a high degree of copy number dynamics, consistent with a birth-death mode of evolution. KAR, SSE, SSZ, and LHS subfamilies evolved in a simple divergent mode with little copy number dynamics. Together, our data revealed that the evolutionary history of this highly conserved and ubiquitous protein family was surprising complex and dynamic.

  3. Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda. (United States)

    Baringou, Stephane; Rouault, Jacques-Deric; Koken, Marcel; Hardivillier, Yann; Hurtado, Luis; Leignel, Vincent


    The 70kDa heat shock proteins (HSP70) are considered the most conserved members of the HSP family. These proteins are primordial to the cell, because of their implications in many cellular pathways (e. g., development, immunity) and also because they minimize the effects of multiple stresses (e. g., temperature, pollutants, salinity, radiations). In the cytosol, two ubiquitous HSP70s with either a constitutive (HSC70) or an inducible (HSP70) expression pattern are found in all metazoan species, encoded by 5 or 6 genes (Drosophila melanogaster or yeast and human respectively). The cytosolic HSP70 protein family is considered a major actor in environmental adaptation, and widely used in ecology as an important biomarker of environmental stress. Nevertheless, the diversity of cytosolic HSP70 remains unclear amongst the Athropoda phylum, especially within decapods. Using 122 new and 311 available sequences, we carried out analyses of the overall cytosolic HSP70 diversity in arthropods (with a focus on decapods) and inferred molecular phylogenies. Overall structural and phylogenetic analyses showed a surprisingly high diversity in cytosolic HSP70 and revealed the existence of several unrecognised groups. All crustacean HSP70 sequences present signature motifs and molecular weights characteristic of non-organellar HSP70, with multiple specific substitutions in the protein sequence. The cytosolic HSP70 family in arthropods appears to be constituted of at least three distinct groups (annotated as A, B and C), which comprise several subdivisions, including both constitutive and inducible forms. Group A is constituted by several classes of Arthropods, while group B and C seem to be specific to Malacostraca and Hexapoda/Chelicerata, respectively. The HSP70 organization appeared much more complex than previously suggested, and far beyond a simple differentiation according to their expression pattern (HSC70 versus HSP70). This study proposes a new classification of cytosolic

  4. Species Identification and Molecular Typing of Leishmania Spp. Using Targeting HSP70 Gene in Suspected Patients of Cutaneous Leishmaniasis from Sistan and Baluchestan Province, Southeast Iran (United States)



    Background: Leishmaniasis is a sand fly-borne disease caused by the protozoan parasites belonging to the genus Leishmania. Because of the preventing and controlling methods, clinical course, prognosis and choice of treatment are differing from species; differentiation of species is critical. The present study was aimed to detect the parasite species using the PCR-RFLP method. Methods: A total of 130 Giemsa-Stained slides from suspected Cutaneous leishmaniasis (CL) patients were examined under a light microscope at ×1000. DNA from each slide was extracted PCR method was undertaken with HSP70 genes and the PCR products were digested with a restriction enzyme HaeIII (BsuR1). The study was conducted in the laboratory of Zahedan University of Medical Sciences in the Sistan and Baluchestan Province, southeastern Iran in 2015. Results: From 130 suspected samples, 59 (45.3%) were positive by the microscopic examination, meanwhile 64 (49.2%) were positive by PCR-RFLP, Leishmania species were recognized, and L. tropica was introduced as predominant species in current study. Conclusion: PCR-RFLP is a valuable technique for distinguish of Leishmania species. Furthermore, anthroponotic CL is the dominant cause of CL in Sistan and Baluchestan Province. PMID:28127360

  5. Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress. (United States)

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


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

  6. Overexpression, Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z Protein.

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    Tawanda Zininga

    Full Text Available Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.

  7. Hsp70 Structure, Function, Regulation and Influence on Yeast Prions


    D. Sharma; Masison, D. C.


    Heat shock proteins protect cells from various conditions of stress. Hsp70, the most ubiquitous and highly conserved Hsp, helps proteins adopt native conformation or regain function after misfolding. Various co-chaperones specify Hsp70 function and broaden its substrate range. We discuss Hsp70 structure and function, regulation by co-factors and influence on propagation of yeast prions.

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

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    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.

  9. Catapult mechanism renders the chaperone action of Hsp70 unidirectional. (United States)

    Gisler, S M; Pierpaoli, E V; Christen, P


    Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins. The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis. We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions. (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1). (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide. For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1. The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2). Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached. This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding. By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated.

  10. Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles. (United States)

    Gehrmann, Mathias K; Kimm, Melanie A; Stangl, Stefan; Schmid, Thomas E; Noël, Peter B; Rummeny, Ernst J; Multhoff, Gabriele


    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.

  11. 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.

  12. Phosphorylation of an envelope-associated Hsp70 homolog in amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.). (United States)

    Checa, S K; Viale, A M


    The presence of Hsp70 and Hsp60 molecular chaperones in amyloplasts isolated from cultured sycamore cells was analyzed by immunoblotting. Hsp70 homologs were located in both amyloplast envelope and stromal fractions, but no Hsp60 homologs were detected in any of the different suborganellar fractions. Incubation of whole amyloplasts or their envelope fraction with Mg2+ gamma-32P-ATP resulted in a rapid phosphorylation of the envelope-associated Hsp70 homolog, which constitutes a major target of phosphorylation in these plastids.

  13. Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET. (United States)

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


    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.

  14. Chemical modulators of heat shock protein 70 (Hsp70) by sequential, microwave-accelerated reactions on solid phase. (United States)

    Wisén, Susanne; Androsavich, John; Evans, Christopher G; Chang, Lyra; Gestwicki, Jason E


    Molecular chaperones, such as Hsp70 and Hsp90, are responsible for a variety of protective, anti-apoptotic functions. While inhibitors of Hsp90, such as geldanamycin and its derivative 17-AAG, are well known and important anti-cancer leads, Hsp70 has received less attention. Interesting lead candidates for Hsp70 share a dihydropyrimidine core; however, the preferred display of pendant functionality is still not clear. Here, we take advantage of the versatility of peptides to explore the requirements for activity. An exploratory compound collection was assembled by performing a Biginelli cyclocondensation at the terminus of a resin-bound beta-peptide. Liberation from solid support yielded peptide-modified dihydropyrimidines and, within this series, we uncovered compounds that alter the ATPase activity of Hsp70 and its bacterial ortholog, DnaK. Moreover, we identified important contributions made by aromatic, hydrophobic groups. These chemical probes could be used to study the roles of this molecular chaperone in disease.

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

    Directory of Open Access Journals (Sweden)

    Gehrmann MK


    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

  16. Structure and expression of a rice hsp70 gene

    Institute of Scientific and Technical Information of China (English)

    王群; 方荣祥


    A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5’ flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5’-CTcgGAAccTTCgAG-3’. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3’-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.

  17. Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones (United States)

    Malinverni, Duccio; Marsili, Simone; Barducci, Alessandro; De Los Rios, Paolo


    Hsp70s are a class of ubiquitous and highly conserved molecular chaperones playing a central role in the regulation of proteostasis in the cell. Hsp70s assist a myriad of cellular processes by binding unfolded or misfolded substrates during a complex biochemical cycle involving large-scale structural rearrangements. Here we show that an analysis of coevolution at the residue level fully captures the characteristic large-scale conformational transitions of this protein family, and predicts an evolutionary conserved–and thus functional–homo-dimeric arrangement. Furthermore, we highlight that the features encoding the Hsp70 dimer are more conserved in bacterial than in eukaryotic sequences, suggesting that the known Hsp70/Hsp110 hetero-dimer is a eukaryotic specialization built on a pre-existing template. PMID:26046683

  18. Inducible HSP70 Protects Radiation-Induced Salivary Gland Damage

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    Lee, Hae-June; Lee, Yoon-Jin; Kwon, Hee-Choong; Lee, Su-Jae; Bae, Sang-Woo; Lee, Yun-Sil [Korea Institute of Radiological Medical Sciences, Seoul (Korea, Republic of); Kim, Sung-Ho [Chonnam National University, Gwangju (Korea, Republic of)


    Irradiation (IR) delivered to the head and neck is a common treatment for malignancies. Salivary glands in the irradiation field are severely damaged, and consequently this resulted in marked salivary hypofunction. While the exact mechanism of salivary gland damage remains enigmatic, fluid secreting acinar cells are lost, and saliva output is dramatically reduced. Previously we have reported that inducible heat shock protein 70 (HSP70i) induced radioresistance in vitro. Moreover, HSP70i localized to salivary glands by gene transfer has great potential for the treatment of salivary gland. Herein, we investigated whether HSP70 can use as radio protective molecules for radiation-induced salivary gland damage in vivo.

  19. The Anti-inflammatory mechanisms of Hsp70

    Directory of Open Access Journals (Sweden)

    Thiago J Borges


    Full Text Available Immune responses to heat shock proteins (Hsp develop in virtually all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, Hsp administration can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory diseases, Hsp peptides have been shown to promote the production of anti-inflammatory cytokines, indicating immunoregulatory potential of Hsp. Therefore, the presence of immune responses to Hsp in inflammatory diseases can be seen as an attempt of the immune system to correct the inflammatory condition. Hsp70 can modulate inflammatory responses in models of arthritis, colitis and graft rejection, and the mechanisms underlying this effect are now being elucidated. Incubation with microbial Hsp70 was seen to induce tolerogenic DCs and to promote a suppressive phenotype in myeloid-derived suppressor cells and monocytes. These DC could induce regulatory T cells (Tregs, independently of the antigens they presented. Some Hsp70 family members are associated with autophagy, leading to a preferential uploading of Hsp70 peptides in MHC class II molecules of stressed cells. Henceforth, conserved Hsp70 peptides may be presented in these situations and constitute targets of Tregs, contributing to downregulation of inflammation. Finally, an interfering effect in multiple intracellular inflammatory signaling pathways is also known for Hsp70. Altogether it seems attractive to use Hsp70, or its derivative peptides, for modulation of inflammation. This is a physiological immunotherapy approach, without the immediate necessity of defining disease specific auto-antigens. In this article, we present the evidence on anti-inflammatory effects of Hsp70 and discuss the need for experiments that will be crucial for the further exploration of the immuno-suppressive potential of this protein.

  20. Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones. (United States)

    Rousaki, Aikaterini; Miyata, Yoshinari; Jinwal, Umesh K; Dickey, Chad A; Gestwicki, Jason E; Zuiderweg, Erik R P


    Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an "allosteric drug." Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.

  1. Heritability of hsp70 expression in the beetle Tenebrio molitor: Ontogenetic and environmental effects. (United States)

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


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

  2. Expression of stress response HSP70 gene in Asian paddle crabs, Charybdis japonica, exposure to endocrine disrupting chemicals, bisphenol A (BPA) and 4-nonylphenol (NP) (United States)

    Park, Kiyun; Kwak, Ihn-Sil


    The Asian paddle crab, Charybdis japonica, is a potential bio-indicator reflecting marine sediment toxicity as well as a commercially important species living along coastal areas in Korea. This study investigated its stress response by looking at the heat shock protein (HSP70) gene of C. japonica when the organism is exposed to bisphenol A (BPA) and 4-nonylphenol (NP). We characterized partial sequence of HSP70 as the stressresponse gene of C. japonica. The nucleotide sequence of C. japonica HSP70 is over 90% homologous with the corresponding gene of other crabs. Phylogenetic tree analysis revealed a close relationship between C. japonica HSP70 and HSP70 in other species of lobster and shrimps. HSP70 mRNA transcripts were detected in all the examined tissues of C. japonica, with the highest level in gills, the organ that most frequently came into contact with the external BPA or NP-laden water. As no reference data were available for C. japonica crab exposure, the BPA and NP 24-h LC50 values have not been previously determined. The expression of the C. japonica HSP70 gene to various BPA or NP concentrations during short and longer times was assessed. Gene expression was significantly induced in concentration- and time-dependent manners after BPA or NP exposures. These results support the postulation that crab C. japonica HSP70 could be a potential stress response molecular marker to monitor marine ecosystems.

  3. Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage. (United States)

    Lee, Byoungchun; Ahn, Younghee; Kang, Sung-Myung; Park, Youngjin; Jeon, You-Jin; Rho, Jong M; Kim, Sung-Woo


    Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.

  4. Molecular cloning of hsf1 and hsbp1 cDNAs, and the expression of hsf1, hsbp1 and hsp70 under heat stress in the sea cucumber Apostichopus japonicus. (United States)

    Xu, Dongxue; Sun, Lina; Liu, Shilin; Zhang, Libin; Yang, Hongsheng


    The heat shock response (HSR) is known for the elevated synthesis of heat shock proteins (HSPs) under heat stress, which is mediated primarily by heat shock factor 1 (HSF1). Heat shock factor binding protein 1 (HSBP1) and feedback control of heat shock protein 70 (HSP70) are major regulators of the activity of HSF1. We obtained full-length cDNA of genes hsf1 and hsbp1 in the sea cucumber Apostichopus japonicus, which are the second available for echinoderm (after Strongylocentrotus purpuratus), and the first available for holothurian. The full-length cDNA of hsf1 was 2208bp, containing a 1326bp open reading frame encoding 441 amino acids. The full-length cDNA of hsbp1 was 2850bp, containing a 225bp open reading frame encoding 74 amino acids. The similarities of A. japonicus HSF1 with other species are low, and much higher similarity identities of A. japonicus HSBP1 were shared. Phylogenetic trees showed that A. japonicus HSF1 and HSBP1 were clustered with sequences from S. purpuratus, and fell into distinct clades with sequences from mollusca, arthropoda and vertebrata. Analysis by real-time PCR showed hsf1 and hsbp1 mRNA was expressed constitutively in all tissues examined. The expression of hsf1, hsbp1 and hsp70 in the intestine at 26°C was time-dependent. The results of this study might provide new insights into the regulation of heat shock response in this species.

  5. Seasonal variation in expression pattern of genes under HSP70 : Seasonal variation in expression pattern of genes under HSP70 family in heat- and cold-adapted goats (Capra hircus). (United States)

    Banerjee, Dipak; Upadhyay, Ramesh C; Chaudhary, Umesh B; Kumar, Ravindra; Singh, Sohanvir; Ashutosh; G, Jagan Mohanarao; Polley, Shamik; Mukherjee, Ayan; Das, Tapan K; De, Sachinandan


    Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.

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

    DEFF Research Database (Denmark)

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


    The cytosolic 70 KDa heat shock proteins (Hsp70s) are widely used as biomarkers of environmental stress in ecological and toxicological studies in fish. Here we analyze teleost genome sequences to show that two genes encoding inducible hsp70s (hsp70-1 and hsp70-2) are likely present in all teleost...... fish. Phylogenetic and synteny analyses indicate that hsp70-1 and hsp70-2 are distinct paralogs that originated prior to the diversification of the teleosts. The promoters of both genes contain a TATA box and conserved heat shock elements (HSEs), but unlike mammalian HSP70s, both genes contain...

  7. Following the Expression of HSP 70 Gene by Semiquantification

    Directory of Open Access Journals (Sweden)

    Jana Tkáčová


    Full Text Available We conducted feeding experiment with broiler chickens Coob 500. We used lucerne meal in the feeding mixtures. It contains carotenoids including lycopen. Lycopene may have important antioxidant implications. Lycopene levels in serum or tissues do not correlate well with the overall intake. It is a potent antioxidant in vitro and in human studies. We followed the effect of lucerne meal on expression of protein HSP 70 in our experiment. HSP 70 belongs to large group of high shock proteins which mediate organism´s response not only to temperature rise, but also to other unfavorable factors, e.g. exposure to heavy metals, hormones, oxidants, etc. We isolated mRNA from heart, spleen, liver, abdominal fat and we transferred transcription and PCR. Expression of HSP 70 was visualized on agarose gel. We detected that the lowest expression was in the heart, the greatest expression was detected in the liver.

  8. Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus. (United States)

    Lamoth, Frédéric; Juvvadi, Praveen R; Soderblom, Erik J; Moseley, M Arthur; Steinbach, William J


    Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus.

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

    Directory of Open Access Journals (Sweden)

    Daniela Pretti da Cunha Tirapelli


    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.

  10. Three Hsp70 genes are located in the C4-H-2D region: Possible candidates for the Orch-1 locus

    Energy Technology Data Exchange (ETDEWEB)

    Snoek, M.; Jansen, M.; Vugt, H. van (The Netherlands Cancer Inst., Amsterdam (Netherlands)); Olavensen, M.G.; Campbell, R.D. (MRC Immunochemistry Univ., Oxford (United Kingdom)); Teuscher, C. (Brigham Young Univ., Provo (United States))


    The central region of the mouse MHC harbors a recombinational hot spot area. Most recombinations in this part of the complex take place between the Hsp70.1 gene and the G7 gene. This interval is of interest since structurally indistinguishable recombinant haplotypes do differ in functional behavior. Susceptibility to experimental allergic orchitis, which is controlled by the Orch-1 locus, is one example. We have analyzed the hot spot region at the molecular level in order to understand the molecular organization of this chromosomal segment. From a C57BL genomic library we constructed a cosmid contig bridging the interval between Hsp70.1 and G7. The Orch-1 gene maps to a 60-kb segment of DNA and which we found a new Hsp70 homologue, Hsp 70.3. Thus, as in the human MHC, the central region of the mouse MHC harbors a cluster of three Hsp70 genes: Hsp 70.1, Hsp 70.3, and Hsc 70t. Two other genes are located in this critical interval (G7b and G7a/Bat-6), and there might still be other undetected genes present in the region. Heat shock proteins play an important role in a large number of physiological processes and it is tempting to speculate that Hcs70t, which exhibits testis-specific expression, may be identical to Orch-1. 32 refs., 2 figs., 2 tabs.

  11. Overproduction and biophysical characterization of human HSP70 proteins. (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Duggan, Kelli D; Tsutsui, Yuko; Hays, Franklin A


    Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.

  12. Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone. (United States)

    Amick, Joseph; Schlanger, Simon E; Wachnowsky, Christine; Moseng, Mitchell A; Emerson, Corey C; Dare, Michelle; Luo, Wen-I; Ithychanda, Sujay S; Nix, Jay C; Cowan, J A; Page, Richard C; Misra, Saurav


    Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

  13. UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii. (United States)

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


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

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

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    Zivar Salehi


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

  15. 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...

  16. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

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    Pedro Fernandez-Funez


    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover

  17. Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity. (United States)

    Ogawa, Kota; Seki, Takahiro; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio


    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins' aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant's degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant's cytotoxicity by enhancing its degradation.

  18. Patterns of variation in levels of hsp70 in natural rocky shore populations from microscales to mesoscales. (United States)

    Halpin, Patricia M; Sorte, Cascade J; Hofmann, Gretchen E; Menge, Bruce A


    An important step in connecting the organismal response to thermal stress to patterns of community structure is determining at what scale discernable levels of variation are manifested. The temperature signal to which organisms may potentially respond varies at many spatial scales including microhabitat, tidal height, site and latitude. A number of studies have taken physiological assessment of the heat shock response (HSR) into the intertidal both as a tool for examining the HSR in nature and for examining the utility of HSR molecules as population or community level indicators. Most commonly, immunodetection of the total pool of the Hsp70 family of isoforms is used. Here we present data on levels of Hsp70 in intertidal organisms from microhabitat to the mesoscale. Our data and previously published work show that Hsp70 levels vary at all scales examined, similar to other physical and biological variables of interest. This demonstrates both the potential utility of Hsp70 detection as a molecular tool for field biologists and to the care that must be taken in assessing scale of variation when looking for potential bioindicator molecules.

  19. Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata. (United States)

    Kalosaka, Katerina; Soumaka, Elisavet; Politis, Nikos; Mintzas, Anastassios C


    The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 degrees C) followed by adult flies and late embryos (100% survival at 39 degrees C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 degrees C increased thermotolerance at higher temperatures by approximately 1 degrees C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.

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

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    Mathias Gehrmann

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

  1. Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress. (United States)

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


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

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

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


    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.

  3. Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

    Institute of Scientific and Technical Information of China (English)

    Ping QU; Yanfang SUI; Libing LIU; Jiahai MA; Guangsheng CHEN; Jiankang CHEN; Fang'e LIU


    To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and puri-fied. DCs were isolated and cultured from human peri-pheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 106 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37℃ for 0.5, 1,2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion pro-tein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluor-escence. The result detected by FCM indicated that after incubation for 0.5 h at 37℃, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37℃, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot exam-ination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09±31.78/105 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36±15.73). There was no significant difference between the 2 groups (P<0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78±1.40)/105 cells and EGFP-loaded (23.13±4,57)/105 cells DC groups in the amount of DCs secreting IL-12 (P<0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs

  4. 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


    We show that inhibition of 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...... activity selectively induces surface expression of Hsp70 on hematopoietic cancer cells and that this may increase immunorecognition of these cells.......-positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis such as etoposide and camptothecin also led to a robust induction of Hsp70 surface expression. Hsp70 expression was, however, not caused by induction of apoptosis per se, as activated CD4 T cells remained Hsp70...

  5. [PCR-RFLP/Hsp70 for identification and tipification of leishmania from the tropical region]. (United States)

    Margarita Montalvo, Ana; Fraga, Jorge; Aylema Romero, Jaqueline; Monzote, Lianet; Ivon Montano, Ing; Dujardin, Jean Claude


    The optimization of the PCR conditions for amplification of the gene coding for the 70 kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, Lamazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosornma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

  6. Genetics of human longevity with emphasis on the relevance of HSP70 as candidate genes. (United States)

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


    Human longevity is determined to a certain extent by genetic factors. Several candidate genes have been studied for their association with human longevity, but the data collected so far are inconclusive. One of the reasons is the choice of the candidate genes in addition to the choice of an appropriate study design and methodology. Since aging is characterized by a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, the focus of studies on human longevity association with genes has now shifted to the pathways of cellular maintenance and repair mechanisms. One such pathway includes the battery of stress response genes, especially the heat shock protein HSP70 genes. Three such genes, HSPA1A, HSPA1B and HSPA1L, are present within the MHC-III region on the short arm of chromosome 6. We and others have found alleles, genotypes and haplotypes which have been significantly associated with human longevity and survival. We have also provided some functional evidence for these genetic associations by showing that isolated peripheral blood cells from those genotypes which are negatively associated with human longevity also have less ability to respond to heat shock. Stress response genes, particularly HSP70, are now the major candidates in the gene-longevity association studies.

  7. Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3.

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    Mathias Gehrmann

    Full Text Available BACKGROUND: Human tumors differ from normal tissues in their capacity to present Hsp70, the major stress-inducible member of the HSP70 family, on their plasma membrane. Membrane Hsp70 has been found to serve as a prognostic indicator of overall patient survival in leukemia, lower rectal and non small cell lung carcinomas. Why tumors, but not normal cells, present Hsp70 on their cell surface and the impact of membrane Hsp70 on cancer progression remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Although Hsp70 has been reported to be associated with cholesterol rich microdomains (CRMs, the partner in the plasma membrane with which Hsp70 interacts has yet to be identified. Herein, global lipid profiling demonstrates that Hsp70 membrane-positive tumors differ from their membrane-negative counterparts by containing significantly higher amounts of globotriaoslyceramide (Gb3, but not of other lipids such as lactosylceramide (LacCer, dodecasaccharideceramide (DoCer, galactosylceramide (GalCer, ceramide (Cer, or the ganglioside GM1. Apart from germinal center B cells, normal tissues are Gb3 membrane-negative. Co-localization of Hsp70 and Gb3 was selectively determined in Gb3 membrane-positive tumor cells, and these cells were also shown to bind soluble Hsp70-FITC protein from outside in a concentration-dependent manner. Given that the latter interaction can be blocked by a Gb3-specific antibody, and that the depletion of globotriaosides from tumors reduces the amount of membrane-bound Hsp70, we propose that Gb3 is a binding partner for Hsp70. The in vitro finding that Hsp70 predominantly binds to artificial liposomes containing Gb3 (PC/SM/Chol/Gb3, 17/45/33/5 confirms that Gb3 is an interaction partner for Hsp70. CONCLUSIONS/SIGNIFICANCE: These data indicate that the presence of Gb3 enables anchorage of Hsp70 in the plasma membrane of tumors and thus they might explain tumor-specific membrane localization of Hsp70.

  8. Hsp70 chaperone systems: diversity of cellular functions and mechanism of action. (United States)

    Mayer, M P; Bukau, B


    Hsp70 chaperone systems play an essential role in the life cycle of many proteins not only in an hostile environment but also under normal growth conditions. In the course of evolution the diversification of functions was accompanied by an amplification of components of the Hsp70 system. Here strategies are reviewed how different Hsp70 systems work independently or cooperate with each other in a functional network to perform their housekeeping tasks even under stress conditions. We further discuss how co-chaperones which act as targeting factors regulate the cycle of substrate binding and release upon which the Hsp70 chaperone activity depends.

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

    Directory of Open Access Journals (Sweden)

    Yoon Jung-Hoon


    Full Text Available Abstract Background Epigallocatechin-3-gallate (EGCG, one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs and tumor suppression. Methods Cell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model. Results Treatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h or oxidative stress (H2O2, 500 μM for 24 h. Moreover, treatment with EGCG (10 mg/kg in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression. Conclusions Overall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  11. Inhibition of α-Synuclein Fibril Elongation by Hsp70 Is Governed by a Kinetic Binding Competition between α-Synuclein Species. (United States)

    Aprile, Francesco A; Arosio, Paolo; Fusco, Giuliana; Chen, Serene W; Kumita, Janet R; Dhulesia, Anne; Tortora, Paolo; Knowles, Tuomas P J; Vendruscolo, Michele; Dobson, Christopher M; Cremades, Nunilo


    The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.

  12. 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

    , membrane-bound Hsp70 can stimulate antigen presenting cells (APCs) to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 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...

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

    DEFF Research Database (Denmark)

    Jensen, Helle

    -derived antigenic peptides, a function which is currently explored in immunotherapeutic approaches against cancer. Additionally, membrane-bound Hsp70 can stimulate antigen presenting cells to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells...... frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...... 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 cell surface expression was confined...

  14. Expression of HSP70 in Peripheral Lymphocytes of the Patients with Allergic Rhinitis

    Institute of Scientific and Technical Information of China (English)

    刘立思; 肖成峰; 张明; 程磊; 王鄂芬; 邬堂春


    The expression levels of heat shock protein 70 (HSP70) from peripheral lymphocytes ofthe patients with allergic rhinitis (AR) and the clinical implication were investigated. In the morn-ing, 3 ml of fasting venous blood was taken out. The lymphocytes were isolated by using Ficoll-Hypaque and the expression of HSP70 in the lymphocytes was detected by using Western blot. Inthe AR patients the HSP70 level (41.49± 15.77 integrated optical density, IOD) were significantlyhigher than that in the control group (23.89±10. 13 IOD, P<0.05). Western blot demonstratedthat HSP70 bands in AR patients were more intensive than those in the control group. It was con-cluded that the elevated HSP70 level in peripheral lymphocytes of the AR patients might contributeto the development of AR.

  15. Dynamic Analysis of the Expression of HSP70 during Experimental Tooth Movement in Rats

    Institute of Scientific and Technical Information of China (English)


    In this study, the expression of HSP70 during experimental tooth movement was dynamically observed and the relationship between HSP70 and orthodontic periodontal tissue remodeling were observed. The orthodontic appliance was placed between the right maxillary first molar and maxillary central incisors of adult SD rats to establish a rat molar movement model. Immunohistochemistry was performed 1, 3, 5, 7 and 14 day(s) after orthodontic force application to observe the expression and localization of inducible HSP70. The expression of HSP70 was strongly positive in the early stage of the tooth movement, became gradually less positive, and was weakly positive in the restoration stage. There was difference in staining pattern between different parts of PDL during the same period. These results suggest that the expression of HSP70 and difference in staining pattern among different parts of PDL during orthodontic tooth movement in rats may be implicated in stress response and remodeling of periodontal tissue.

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

    DEFF Research Database (Denmark)

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


    Expression of heat-inducible Hsp70 is considered closely linked to thermotolerance in Drosophila melanogaster and other ectotherms. However, intra-specific variation of Hsp70 expression levels and its relationship to heat resistance has only been investigated in a few studies. Although...... in adult heat tolerance. Therefore, although Hsp70 expression is a major component of the cellular heat stress response, its influence on intra-specific heat tolerance variation may be life-stage specific. J. Exp. Zool. 313A:35-44, 2010. © 2009 Wiley-Liss, Inc...... in Drosophila larvae Hsp70 expression may be a key determinant of heat tolerance, the evidence for this in adults is equivocal. We therefore examined heat-induced Hsp70 expression and several measurements of adult heat tolerance in three independent collections of D. melanogaster, measured in three laboratories...

  17. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters. (United States)

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I


    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  18. Could Upregulated Hsp70 Protein Compensate for the Hsp90-Silence-Induced Cell Death in Glioma Cells?

    Directory of Open Access Journals (Sweden)

    Chinmay Munje


    Full Text Available The molecular chaperone heat shock protein 90 alpha (Hsp90α has been recognized in various tumours including glioma. This pilot study using a proteomic approach analyses the downstream effects of Hsp90 inhibition using 17-allylamino-17-demethoxygeldanamycin (17AAG and a short hairpin RNA (shRNA oligonucleotide targeting hsp90α (shhsp90α in the U87-MG glioma cell line. Preliminary data coupled with bioinformatic analysis identified several known and unknown Hsp90 client proteins that demonstrated a change in their protein expression after Hsp90 inhibition, signifying an alteration in the canonical pathways of cell cycle progression, apoptosis, cell invasion, angiogenesis, and metastasis. Members of the glycolysis pathway were upregulated, demonstrating increased dependency on glycolysis for energy source by the treated glioma cells. Upregulated proteins also include Hsp70 and members of its family such as Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function after Hsp90 inhibition. Considering Hsp70’s role in antiapoptosis, it was postulated that a combination therapy involving a multitarget approach could be carried out. Consequently inhibition of both Hsp90 and Hsp70 in U87-MG glioma cells resulted in 60% cell death indicating the importance of combination therapy for glioma therapeutics.

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

    DEFF Research Database (Denmark)

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


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

  20. Glial Hsp70 Protects K+ Homeostasis in the Drosophila Brain during Repetitive Anoxic Depolarization (United States)

    Armstrong, Gary A. B.; Xiao, Chengfeng; Krill, Jennifer L.; Seroude, Laurent; Dawson-Scully, Ken; Robertson, R. Meldrum


    Neural tissue is particularly vulnerable to metabolic stress and loss of ion homeostasis. Repetitive stress generally leads to more permanent dysfunction but the mechanisms underlying this progression are poorly understood. We investigated the effects of energetic compromise in Drosophila by targeting the Na+/K+-ATPase. Acute ouabain treatment of intact flies resulted in subsequent repetitive comas that led to death and were associated with transient loss of K+ homeostasis in the brain. Heat shock pre-conditioned flies were resistant to ouabain treatment. To control the timing of repeated loss of ion homeostasis we subjected flies to repetitive anoxia while recording extracellular [K+] in the brain. We show that targeted expression of the chaperone protein Hsp70 in glial cells delays a permanent loss of ion homeostasis associated with repetitive anoxic stress and suggest that this is a useful model for investigating molecular mechanisms of neuroprotection. PMID:22174942

  1. Glial Hsp70 protects K+ homeostasis in the Drosophila brain during repetitive anoxic depolarization.

    Directory of Open Access Journals (Sweden)

    Gary A B Armstrong

    Full Text Available Neural tissue is particularly vulnerable to metabolic stress and loss of ion homeostasis. Repetitive stress generally leads to more permanent dysfunction but the mechanisms underlying this progression are poorly understood. We investigated the effects of energetic compromise in Drosophila by targeting the Na(+/K(+-ATPase. Acute ouabain treatment of intact flies resulted in subsequent repetitive comas that led to death and were associated with transient loss of K(+ homeostasis in the brain. Heat shock pre-conditioned flies were resistant to ouabain treatment. To control the timing of repeated loss of ion homeostasis we subjected flies to repetitive anoxia while recording extracellular [K(+] in the brain. We show that targeted expression of the chaperone protein Hsp70 in glial cells delays a permanent loss of ion homeostasis associated with repetitive anoxic stress and suggest that this is a useful model for investigating molecular mechanisms of neuroprotection.

  2. Ste20-like kinase, SLK, activates the heat shock factor 1 - Hsp70 pathway. (United States)

    Cybulsky, Andrey V; Guillemette, Julie; Papillon, Joan


    Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-μ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury.

  3. Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model. (United States)

    Yu, Wen-Ying; Chuang, Tien-Fu; Guichard, Cécile; El-Garch, Hanane; Tierny, Dominique; Laio, Albert Taiching; Lin, Ching-Si; Chiou, Kuo-Hao; Tsai, Cheng-Long; Liu, Chen-Hsuan; Li, Wen-Chiuan; Fischer, Laurent; Chu, Rea-Min


    Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true

  4. Hsp70 vaccination-induced primary immune responses in efferent lymph of the draining lymph node. (United States)

    Vrieling, Manouk; Santema, Wiebren; Vordermeier, Martin; Rutten, Victor; Koets, Ad


    Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis (MAP). In earlier studies we showed the protective effect of Hsp70/DDA subunit vaccination against paratuberculosis. In the current study we set out to measure primary immune responses generated at the site of Hsp70 vaccination. Lymph vessel cannulation was performed to obtain efferent lymph from the prescapular lymph node draining the neck area where the vaccine was applied. Hsp70 vaccination induced a significant increase of CD21(+) B cells in efferent lymph, accounting for up to 40% of efferent cells post-vaccination. Proliferation (Ki67(+)) within the CD21(+) B cell and CD4(+) T cell populations peaked between day 3 and day 5 post-vaccination. From day 7, Hsp70-specific antibody secreting cells (ASCs) could be detected in efferent lymph. Hsp70-specific antibodies, mainly of the IgG1 isotype, were also detected from this time point onwards. However, post-vaccination IFN-γ production in efferent lymph was non-sustained. In conclusion, Hsp70-vaccination induces only limited Th1 type immune responsiveness as reflected in efferent lymph draining the vaccination site. This is in line with our previous observations in peripheral blood. The main primary immunological outcome of the Hsp70/DDA subunit vaccination is B cell activation and abundant Hsp70-specific IgG1 production. This warrants the question whether Hsp70-specific antibodies contribute to the observed protective effect of Hsp70 vaccination in calves.

  5. Hsp70 as an indicator of stress in the cells after contact with nanoparticles (United States)

    Hardilová, Šárka; Havrdová, Markéta; Panáček, Aleš; Kvítek, Libor; Zbořil, Radek


    In recent years, production of nanoparticles is increased and thus grows our contact with them too. Question of safety is closely related to the issue of use nanoparticles. There are a number of tests that monitor the viability, ROS production, the effect on the DNA and cell cycle, however, rarely encountered studies on stress in the cells after contact with nanoparticles. Heat shock proteins (HSP) are among the substances that can be used for monitoring stress in cells. HSP are structures with a chaperone activity. They are evolutionarily very old, conservative and they are found with a high degree of homology in prokaryotes and eukaryotes including humans. They exist at low concentrations under physiological conditions, while in the denaturing conditions e.g. high or low temperature, radiation, exposure to chemicals, heavy metals, or nanoparticles their expression is changed. HSPs are involved in maintaining homeostasis in the cell that the denatured protein conformations allow recovery to the original stage. One of the most common proteins from HSP family is Hsp70 - protein with a molecular weight of 70 kDa. The level of Hsp70 in a cell after exposure to the stress changes depending on the stress level to which the cell is exposed to and a time period during which lasted stressful conditions. Our research monitors stress levels of cells manifesting by Hsp70 production after contact with silver nanoparticles. Nanoparticles show different toxicity towards different types of target cells, which is reflected in the values of IC50 - concentration that kills 50% tested cells. Concentration of test substance toxic to one cell type may be innocuous to cells of another type. IC50 obtained from the MTT assay provides a suitable default data and if multiples of IC50 values are used, we can compare and generalize. Studies can be used to compare stress levels in cells that show different sensitivity to the tested nanoparticles compared with cells under optimal growth

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

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


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

  7. Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-hui; TANG Hong-tai; LU Jian; XIA Zhao-fan


    Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.

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

    Directory of Open Access Journals (Sweden)

    Federica Chiappori

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

  9. In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis and rice as putative co-chaperones of Hsp90/Hsp70.

    Directory of Open Access Journals (Sweden)

    Bishun D Prasad

    Full Text Available The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a 'carboxylate clamp'. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1, DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.

  10. Study on the Preparation of Recombinant Human HSP70 and Its Presenting-Antigen Function

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Guimei(


    [1]Yasuaki T Norioki T Noriyuki S et al.70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host's anti-tumor immunity.J Immunol 1993 151:5516[2]Yasuaki T Ping P Kang L et al.Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science 1997 278:117[3]Heiichiro V Pramod K S.Heat shock protein 70-associated peptides elicit specific cancer immunity.J Exp Med 1998 178:1391[4]Sambrook J Fritch E F Maniatis T.Molecular Cloning a laboratory manu 2nd ed.New York:Cold Spring Harbor Laboratory Press 1989.253[5]Satish J Peter M Stanley R et al.Human stress protein hsp70:overexpression in E.coli purification and characterization.Bio/Technology 1995 113:1105[6]哈密斯B.D. 利克伍德D.著.刘毓秀 程桂芳译.蛋白质的凝胶电泳实践方法.北京:科学出版社 1994.151[7]Anne-marie T C Max P Carey L O et al.Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes.J Exp Med 1998 187:685[8]Nandan D Daubenberger C Mpimbaza G.A rapid single-step purification method for immunogenic members of the Hsp70 family:validation and application.J Immunol Methods 1994 176(2) :255

  11. Expression and Significance of HSP70 and MGMT in Breast Neoplasms%HSP70和MGMT在乳腺肿瘤中的表达和意义

    Institute of Scientific and Technical Information of China (English)

    吴荣薇; 薄爱华; 王发亮; 芦广萍


    目的:探讨乳腺肿瘤组织中热休克蛋白HSP70和DNA修复酶O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methyguanine-DNA methytransferase,MGMT)的表达及临床意义.方法:采用免疫组织化学SABC法检测65例乳腺肿瘤组织中HSP70和MGMT的表达情况.结果:乳腺肿瘤中HSP70的阳性率为80.00%(52/65),MGMT的阳性率为60.00%(39/65),二者呈中度相关性(r=0.46).其中,有淋巴结转移组的阳性率高于无转移组(P<0.05).结论:HSP70和MGMT的表达率与淋巴结转移情况有关.检测HSP70与MGMT对乳腺肿瘤预后和化疗方案的选择有指导意义.

  12. Polymorphism analysis of the hsp70 stress gene in Broiler chickens (Gallus gallus of different breeds

    Directory of Open Access Journals (Sweden)

    Carmen Maria Mazzi


    Full Text Available The promoter region and the beginning of the coding region of the hsp70 stress gene were analysed in broiler chickens of a commercial breed (Hubbard-Pettersen, a breed selected for weight gain (PP1 and a non-selected breed (naked-neck Label Rouge. The naked neck gene (Naked neck, Na, which reduces feathering in birds and is thus related to heat resistance, was present in both PP1 and Label Rouge breeds. Genomic DNA was restricted with PstI and Southern blotting analysis of the samples revealed the absence of polymorphic sites for that enzyme in the promoter region and beginning of the coding region of the hsp70 gene of studied birds. PCR-SSCP analysis of these regions, however, indicated the presence of polymorphisms in the beginning of the coding region and the sequencing of the PCR products confirmed and identified two polymorphic sites in this region: a transition A ® G in position +258 and a transversion C ® G in position +276. Both mutations were considered to be silent, since they did not modify the aminoacid sequence of the protein Hsp70. The promoter region of the hsp70 gene was identical in all studied birds, indicating that the regulation pattern of this gene must be the same in all birds at the promoter region. Three different alleles (hsp70-1, hsp70-2 and hsp70-3 were identified for the hsp70 gene from the observed mutations. The allele hsp70-3 was detected in only two breeds, Hubbard-Pettersen and PP1, but at a low frequency (0,016 and 0,006, respectively.

  13. Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation*


    Wang, Adrienne M; Morishima, Yoshihiro; Clapp, Kelly M.; Peng, Hwei-Ming; Pratt, William B.; Gestwicki, Jason E.; Osawa, Yoichi; Lieberman, Andrew P.


    The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well est...

  14. Study on HSP70 Gene Expression in Different Tissue of Cyprinus carpio%鲤鱼HSP70基因组织表达差异研究

    Institute of Scientific and Technical Information of China (English)

    林亚秋; 李瑞文


    [Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cyprinus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio (AY120894), one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cyprinus carpio HSP70 gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 96% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cyprinus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them (P>0.05). There was no significant difference among the expression in three tissues of intestine, mucus and fat (P>0.05), but their expression was significantly higher than those in gonad and gill (P<0.05). [Conclusion] HSP70 gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.

  15. Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70B' in human keratinocytes modulating the proinflammatory response. (United States)

    Buommino, Elisabetta; Schiraldi, Chiara; Baroni, Adone; Paoletti, Iole; Lamberti, Monica; De Rosa, Mario; Tufano, Maria Antonietta


    The heat shock proteins (Hsps) have an important role in the cytoprotection and repair of cells and tissues. One potential mechanism of protection is the ability of Hsp to inhibit genetic expression of proinflammatory cytokines, the transcription of which is dependent on nuclear factor-kappa B (NF-kappaB) activation. In this study, we evaluated the ability of ectoine, a novel natural biomolecule produced by halophilic microorganisms, to activate the hsp70 and hsp70B'. By reverse transcriptase-polymerase chain reaction and Western blot analysis, we demonstrated increased hsp70B' gene expression in human keratinocytes treated with ectoine and heat stressed. In contrast, in the absence of heat shock, ectoine was unable to induce hsp70B' but had the ability to induce another member of the Hsp family, the hsp70. The latter is not only elevated in response to stress but is also present at basal level in unstressed cells. In addition, ectoine had no effect on proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor-alpha and on NF-kappaB and IkappaB-alpha pathway, whereas it downregulated the expression of cited proinflammatory cytokines, in lipopolysaccharides-treated keratinocytes. These results highlighted the ability of ectoine to protect cells from stress conditions and to prevent cell damage by maintaining an elevated level of the Hsp70. Overall, these data might suggest the use of this compatible solute in cosmetic and even pharmaceutical preparations aiming to activate a cytoprotective heat shock response in human cells.

  16. Changing of the HSP70 Content in the Baikal Endemic Sponges Lubomirskiidae Under Conditions of Hyperthermia

    Directory of Open Access Journals (Sweden)

    Itskovich V.B.


    Full Text Available Baikal endemic sponges (Lubomirskiidae make up the bulk of the benthos biomass of the lake. For the first time the changes in the content of HSP70 in response to elevated environment temperature were analyzed in three endemic species of Baikal sponges: Baikalospongia bacillifera (Dybowski, 1880, B. intermedia (Dybowski, 1880 and Swartschewskia papyracea (Dybowski, 1880. Interspecific variability of constitutive HSP70 level was revealed for representatives of the three analyzed Lubomirskiidae species. After exposure at 13 °С for 3 and 7 days opposite changes were noted in the amount of HSP70. Under conditions of hyperthermia the protein level decrease at Baikalospongia species, while at the S. papyracea HSP70 content slightly increased. The differences in the mechanisms of stress adaptation probably affect the thermal resistance of the species, as well as are evidence supporting their specific status.

  17. Variation in Hsp70-1A Expression Contributes to Skin Color Diversity. (United States)

    Murase, Daiki; Hachiya, Akira; Fullenkamp, Rachel; Beck, Anita; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori; Manga, Prashiela


    The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes, and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either white or African American skin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that heat shock protein 70-1A (Hsp70-1A) protein levels were significantly higher in African American melanocytes compared with white melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70 family members in tyrosinase post-translational modification. In addition, pharmacologic inhibition and small interfering RNA-mediated downregulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes, and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.

  18. Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop). (United States)

    Gitau, Grace W; Mandal, Pradipta; Blatch, Gregory L; Przyborski, Jude; Shonhai, Addmore


    Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.

  19. Expression of Hsp70 and Caspase-3 in rabbits after severe traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jing; TAO Dai-qin; ZHAO Hui; YIN Zhi-yong


    Objective:To investigate the expression of Caspase-3 and Hsp70 in rabbits after severe traumatic brain injury (TBI) and to explore the feasibility of its application in estimation of injury time in forensic medicine.Methods:A rabbit model of heavy TBI was developed by high velocity impact on the parietal bone with an iron stick.Totally 8 healthy adult New Zealand white rabbits were randomly divided into control group (n=2) and injury group (n=6).Four hours after injury,tissue specimens from the parietal lobe,temporal lobe,occipital lobe,cerebellum and brainstem were harvested to detect the expression of Hsp70 and Caspase-3 by immunohistochemistry.Besides,the gray values of cells positive for Hsp70 and Caspase-3 were analyzed with an image analyzer.Results:Immunohistochemistry staining demonstrated a low level of Caspase-3 and Hsp70 expression in normal control group.While in injury group,both the Caspase-3and Hsp70 expression was significantly elevated (P<0.05).Positive cells gathered around the lesion focus.Occipital lobe and cerebellum had fewer positive cells while temporal and brainstem had the fewest.Conclusion:The expression of Caspase-3 and Hsp70 at an early stage following severe TBI is characteristic and can be applied to estimate the time of injury.

  20. Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle (United States)

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


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

  1. Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids. (United States)

    Kumar, Jaideep; Chaudhury, Ashok; Bera, Bidhan C; Kumar, Ritesh; Kumar, Rajender; Tatu, Utpal; Yadav, Suresh Chandra


    The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

  2. Research Progress of HSP70 and Its Effects in Exercises%热休克蛋白70及其在运动中作用研究进展

    Institute of Scientific and Technical Information of China (English)

    郑妩媚; 王福文


    Heat shock protein 70 (HSP70) is a molecular chaperone that is highly expressed in response to stress and closely related to exercises. HSP70 plays an important role in body movement, and is considered as a new endogenous protection approach for myocardium in recent years. The research progress in biological characteristics of HSP70 and its protective effect on myocardial damage induced by exercises is reviewed in this paper, to provide basis for reasonable training in exercises.%热休克蛋白70(heat shock protein 70,HSP70)是一种在应激条件下诱导高表达的伴侣蛋白,与运动关系密切,在机体运动过程中起重要作用。近年,HSP70已成为较受重视的心肌内源性保护途径之一。本文主要介绍了HSP70的生物学特性及其对运动性心肌损伤保护作用的研究进展,以冀为合理进行运动训练提供依据。

  3. Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein (United States)

    Rodina, Anna; Patel, Pallav D.; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J.H.; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R.; Patel, Hardik J.; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T.; Young, Jason C.; Chiosis, Gabriela


    SUMMARY Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  4. Cloning and Sequence Analysis of Hsp90 and Hsp70 Gene from Carposina sasakii Matsumura under Heat Stress%高温胁迫下桃小食心虫热激蛋白Hsp90和Hsp70基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    高萍; 贾向风; 王洪平


    桃小食心虫(Carposina sasakii Matsumura)是果树上重要的食心虫类害虫,热激蛋白Hsp90和Hsp70在昆虫抵御温度胁迫反应中具有重要作用。采用RT-PCR方法克隆获得了高温胁迫下桃小食心虫热激蛋白Hsp90和Hsp70基因cDNA部分序列,并对其进行分析,为深入揭示桃小食心虫对环境适应的分子机理提供理论依据。已获得的桃小食心虫热激蛋白Hsp90基因(GenBank登录号:KJ139642)序列长420bp,编码139个氨基酸残基,推导的氨基酸序列中含有热激蛋白90家族的一段序列为YSNKEIFLRE的特征序列,并且cDNA序列在N端具有Hsp90基因保守的ATPase结构,该序列与天蚕(Antheraea yamamai)和柞蚕(Antheraea pernyi)等昆虫的氨基酸序列一致性高达99%。已获得的Hsp70-1基因(GenBank登录号:KJ139643)和Hsp70-2基因(GenBank登录号:KJ139644)序列长均为305bp,编码101个氨基酸残基。 Hsp70-1基因推导的氨基酸序列与家蚕(Bombyx mori)的氨基酸序列一致性为94%,Hsp70-2基因推导的氨基酸序列与小菜蛾(Plutella xylostella)和烟草夜蛾(Manduca sexta)等昆虫的氨基酸序列一致性为96%。%Peach fruit moth,Carposina sasakii Matsumura is one of the main pests of the fruit trees. The heat shock protein 90 (Hsp90)and the heat shock protein70(Hsp70)widely exist in insects and play important role under temperature stress. In order to provide theoretical basis for exploring molecular mechanism of insect adaption to environment, partial-length cDNA encoding Hsp90 and Hsp70 from Carposina Sasakii were cloned by RT-PCR and the sequence was analyzed. Sequence analysis indicated that the partial cDNA of Hsp90 (GenBank accession number: KJ139642) was 420bp, encoding 139 amino acid residues. The deduced amino acid sequence contains an important Hsp90 family characteristic sequence(YSNKEIFLRE) and conservative ATPase structure in N-terminal domain, which shares 99% amino acid sequence identies with the Hsp90 genes from

  5. Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1) (United States)

    Shevtsov, Maxim A.; Nikolaev, Boris P.; Ryzhov, Vyacheslav A.; Yakovleva, Ludmila Y.; Marchenko, Yaroslav Y.; Parr, Marina A.; Rolich, Valerij I.; Mikhrina, Anastasiya L.; Dobrodumov, Anatolii V.; Pitkin, Emil; Multhoff, Gabriele


    The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T2-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M2 measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy

  6. Induction of heat shock protein 70 (Hsp70 prevents neuregulin-induced demyelination by enhancing the proteasomal clearance of c-Jun

    Directory of Open Access Journals (Sweden)

    Rick T Dobrowsky


    Full Text Available Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy and possibly, demyelinating neuropathies. 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-ylacetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90 and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell-DRG (dorsal root ganglia sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1. Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type and Hsp70 KO (knockout mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.

  7. Induction of heat shock protein 70 (Hsp70) prevents neuregulin-induced demyelination by enhancing the proteasomal clearance of c-Jun. (United States)

    Li, Chengyuan; Ma, Jiacheng; Zhao, Huiping; Blagg, Brian S J; Dobrowsky, Rick T


    Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy) and possibly, demyelinating neuropathies. 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] is a small molecule inhibitor of Hsp90 (heat shock protein 90) and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell)-DRG (dorsal root ganglia) sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1). Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase) and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type) and Hsp70 KO (knockout) mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.

  8. Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats. (United States)

    Choudhary, Arbind Kumar; Devi, Rathinasamy Sheela


    Aspartame, a "first generation sweetener", is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake (ADI) value of aspartame to be 50 mg/kg·day, while the JECFA (Joint FAO/WHO Expert Committee on Food Additives) has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/(kg·day)] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period (90-days) has any effect on the expression of hsp70, bcl-2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes.

  9. Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats (United States)

    Choudhary, Arbind Kumar; Devi, Rathinasamy Sheela


    Abstract Aspartame, a “first generation sweetener”, is widely used in a variety of foods, beverages, and medicine. The FDA has determined the acceptable daily intake (ADI) value of aspartame to be 50 mg/kg·day, while the JECFA (Joint FAO/WHO Expert Committee on Food Additives) has set this value at 40 mg/kg of body weight/day. Safety issues have been raised about aspartame due to its metabolites, specifically toxicity from methanol and/or its systemic metabolites formaldehyde and formic acid. The immune system is now recognized as a target organ for many xenobiotics, such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of apoptosis. Our previous studies has shown that oral administration of aspartame [40 mg/(kg·day)] or its metabolites for 90 days increased oxidative stress in immune organs of Wistar albino rats. In this present study, we aimed to clarify whether aspartame consumption over a longer period (90-days) has any effect on the expression of hsp70, bcl-2 and bax at both mRNA transcript and protein expression levels in immune organs. We observed that oral administration of aspartame for 90 days did not cause any apparent DNA fragmentation in immune organs of aspartame treated animals; however, there was a significant increase in hsp70 expression, apart from significant alteration in bcl-2 and bax at both mRNA transcript and protein expression level in the immune organs of aspartame treated animals compared to controls. Hence, the results indicated that hsp70 levels increased in response to oxidative injury induced by aspartame metabolites; however, these metabolites did not induce apoptosis in the immune organs. Furthermore, detailed analyses are needed to elucidate the precise molecular mechanisms involved in these changes.

  10. Gene expression profiles of cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response to thermal stress: possible implications for coral bleaching. (United States)

    Rosic, Nedeljka N; Pernice, Mathieu; Dove, Sophie; Dunn, Simon; Hoegh-Guldberg, Ove


    Unicellular photosynthetic dinoflagellates of the genus Symbiodinium are the most common endosymbionts of reef-building scleractinian corals, living in a symbiotic partnership known to be highly susceptible to environmental changes such as hyperthermic stress. In this study, we identified members of two major heat shock proteins (HSPs) families, Hsp70 and Hsp90, in Symbiodinium sp. (clade C) with full-length sequences that showed the highest similarity and evolutionary relationship with other known HSPs from dinoflagellate protists. Regulation of HSPs gene expression was examined in samples of the scleractinian coral Acropora millepora subjected to elevated temperatures progressively over 18 h (fast) and 120 h (gradual thermal stress). Moderate to severe heat stress at 26°C and 29°C (+3°C and +6°C above average sea temperature) resulted in an increase in algal Hsp70 gene expression from 39% to 57%, while extreme heat stress (+9°C) reduced Hsp70 transcript abundance by 60% (after 18 h) and 70% (after 120 h). Elevated temperatures decreased an Hsp90 expression under both rapid and gradual heat stress scenarios. Comparable Hsp70 and Hsp90 gene expression patterns were observed in Symbiodinium cultures and in hospite, indicating their independent regulation from the host. Differential gene expression profiles observed for Hsp70 and Hsp90 suggests diverse roles of these molecular chaperones during heat stress response. Reduced expression of the Hsp90 gene under heat stress can indicate a reduced role in inhibiting the heat shock transcription factor which may lead to activation of heat-inducible genes and heat acclimation.

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

    Directory of Open Access Journals (Sweden)

    G. Tomasello


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

  12. Influence of induced heat stress on HSP70 in buffalo lymphocytes. (United States)

    Mishra, A; Hooda, O K; Singh, G; Meur, S K


    Heat stress in farm animals, such as cattle and buffalo during summer and post-summer seasons is a problem for livestock producers. The effect of heat stress becomes pronounced when heat stress is accompanied with ambient humidity impairing the immune status, growth, production and reproductive performance of animals. Increase in HSP70 levels from cell cultures in presence of different stressors often does not reflect the physiological adaptability of animals governing thermal regulation. In this study we directly compared the effect of different heat stress conditions with the immune status and HSP70 expression patterns from buffalo lymphocytes both in vivo and in vitro. Murrah buffalo calves were exposed to induced heat stress with two experimental treatments: hot-dry (42 °C with existing relative humidity) or hot humid (35 °C with 70% relative humidity) condition in psychometric chamber, 4 h daily for 12 days and compared with control animals maintained in an experimental shed under natural conditions. There was >200-fold increase in serum-HSP70 levels in both heat stress conditions compared with control. Furthermore, the immune status of the calves failed to activate the level of HSP70 expression in serum lymphocytes. Lymphocytes cultured in vitro at higher temperature exert 2.5-fold increase in HSP70 concentration. This study is the first of its kind to demonstrate more complex expression pattern of buffalo serum-HSP70 level as a thermo adaptive response compared with in vitro treated cells. Results from this study indicate that serum-HSP70 levels could be used as a sensitive biomarker for heat stress management in large farm animals.

  13. Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo (United States)

    Kruse, D. E.; Mackanos, M. A.; O'Connell-Rodwell, C. E.; Contag, C. H.; Ferrara, K. W.


    The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm-2 up to 352 W cm-2. The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

  14. Sequence Analysis of HSP70 Gene of Leishmania major and Leishmania tropica in Chabahar and Mashhad

    Directory of Open Access Journals (Sweden)

    Mansour Dabirzadeh


    Full Text Available Background and Objective: Cutaneous leishmaniasis is a parasitic disease and a health problem in different parts of Iran, especially two cities of Mashhad and Chabahar. Due to morphological similarities of most Leishmania species and difference in reservoirs of L. major and L. tropica, it is necessary to determine the parasite specie to combat the disease. Thus, this study used gene sequencing and genotyping of 70-kDa heat shock protein (HSP70 to differentiate the two species of Leishmania. Methods: In this descriptive-analytical study, microscope slides and cultures were prepared from 43 patients suspected of cutaneous leishmaniasis in Chabahar and Mashhad. PCR was performed after genomic DNA extraction and then PCR products were sequenced and analyzed. Results: Of the 43 patients studied, 32 direct smear and culture (74.4% were positive and 11 (25.6% showed negative results, and were therefore excluded from the study. Using HSP70-specific primers, 1962 bp and 1152bp bands were observed for HSP70 of L. major in Chabahar and L. tropica in Mashhad, respectively. Based on the results, there were 18 nucleotide differences between HSP70 of L. major in Chabahar and L. tropica in Mashhad. Conclusion: Due to the morphological similarities between Leishmania species and inability to differentiate species through parasitological methods, the HSP70 gene can be used for identification of the species, and prevention and treatment of the disease.

  15. Changes of levels of hs-CRP, Hsp70, and MPO in patients with preeclampsia

    Institute of Scientific and Technical Information of China (English)

    Xia Liu


    Objective:To observe the changes of the levels of hs-CRP, Hsp70, and MPO in patients with preeclampsia (PE) and their clinical significance. Methods:A total of 60 pregnant women with mild and severe PE who were admitted in our hospital from January, 2013 to March, 2014 were included in the study and divided into the mild group and the severe group with 30 cases in each group. The levels of serum hs-CRP, Hsp70, and MPO were detected, and their relations with PE were analyzed. The lipid metabolism related indicators were meanwhile detected. A total of 30 healthy later pregnant women at the same stage were served as the control group for comparative analysis. Results:The levels of serum hs-CRP, Hsp70, and MPO in the mild and severe PE group were significantly higher than those in the control group (P0.05). Conclusions: The abnormal lipid metabolism in different degrees exists in the pregnant women with PE, and hs-CRP, Hsp70, and MPO are together involved in the pathogenesis of PE. Detection of the levels of hs-CRP, Hsp70, and MPO in the pregnant women with PE can accurately reflect the condition of PE.

  16. Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection. (United States)

    Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N; Gestwicki, Jason E; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith


    Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.

  17. 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:; 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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Asita Elengoe


    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.

  19. Detection of constitutive and inducible HSP70 proteins in formalin fixed human brain tissue. (United States)

    Preusse-Prange, A; Modrow, J-H; Schwark, T; von Wurmb-Schwark, N


    The investigation of formalin fixed and paraffin embedded tissue is a routine method in forensic histology. Since these samples are usually stored for decades they provide a unique tissue bank for different scientific issues. In the past, numerous studies were conducted using different kinds of paraffin embedded tissues. However, it is well known that formalin affects macromolecules and thus might hamper reliable and reproducible molecular experiments. The aim of this study was to find out if the treatment with formalin has a negative effect on different protein detection methods and additionally to define the dimension of those possible deleterious effects. We incubated brain tissue samples in formalin for up to three months. After incubation, the samples were analyzed using immunohistochemistry (IHC) and Western blotting to specifically detect and quantify members of the HSP70 superfamily (heat shock proteins). Our study shows that the Western blot analysis of formalin fixed tissues does not allow a reliable detection of proteins at all, while a reproducible detection by IHC was still possible after one month of incubation.

  20. Protective Effects of Overexpression TCR Vβ5.2-HSP70 and TCR Vβ8.2-HSP70 against Collagen-Induced Arthritis in Rats

    Institute of Scientific and Technical Information of China (English)

    Jing Xiao; Shentao Li; Wei Wang; Yun Li; Wenming Zhao


    Collagen-induced arthritis (CIA) is an animal model, which closely resembles human rheumatoid arthritis (RA) in pathogenesis and pathology. Evidence suggests that the inhibition of T lymphocytes or their functions can alleviate the progression of arthritis. So the administration of arthritogenic T cell receptor (TCR) variable region peptide or DNA vaccines encoding pathogenic TCR Vβ variable region may provide useful information for designing specific immunotherapies against autoimmune diseases. Heat shock proteins (HSPs) have the function of raising antigenic immunogenicity and HSP70 has a protective effect against arthritis. We previously demonstrated the presence of pathogenic predominant T cell receptor Vβ5.2 and Vβ8.2 clonotypes in the joints of CIA rats. In this study, we constructed the recombinant eukaryotic expression vectors pTARGET-TCR Vβ5.2/8.2-HSP70, and evaluated their protective effects on CIA rats. Protective effects were observed in CIA rats by injecting these recombinant DNA vaccines, which could alleviate arthritis index, decrease the levels of IFN-γ and anti-CⅡ antibody in serum, and increase the levels of IL-4. Pathological changes were not as serious as those observed in control CIA rats. The rat injected with two combined vaccines showed better protective effects than CIA rats administered with individual vaccine. These results showed that recombinant DNA vaccines pTARGET-TCR Vβ5.2-HSP70 and pTARGET-TCR Vβ8.2-HSP70 could significantly alleviate the arthritic symptoms of CIA rats, and better protective effects could be achieved if these two vaccines were used in combination.

  1. Cloning and characterization of a heat shock protein 70 gene, MsHSP70-1, in Medicago sativa

    Institute of Scientific and Technical Information of China (English)

    Zhishui He; Rong Xie; Yanzhang Wang; Huasong Zou; Jiabi Zhu; Guanqiao Yu


    Some members of the heat shock protein 70 (HSP70) family have important functions in organism development. Here, we identified an alfalfa (Medicago sativa L. cv. Algonquin) HSP70 gene, MsHSP70-1, using cDNA array and reverse transcription-polymerase chain reaction. This gene contains a 1947 bp open reading frame encoding a protein of 649 amino acids.This protein contains all conserved domains,motifs.and characteristic sequences of plant HSP70s.The expression of MsHsP70-1 is enhanced in nodule compared withroot,stem,leaf,and flower,and throughout the process of nodule development.Northern hybridization analysis indicated that the expression of MsHSP70-1 in nodule requires the active bacA gene of rhizobia.These results suggested that MsHSP70-1 might play an important role in alfalfa nodule development.

  2. Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells. (United States)

    Kust, Nadezda; Rybalkina, Ekaterina; Mertsalov, Ilya; Savchenko, Ekaterina; Revishchin, Alexander; Pavlova, Gali


    The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

  3. Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity. (United States)

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


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

  4. Analogs of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, as Anti-Cancer Agents. (United States)

    Li, Xiaokai; Srinivasan, Sharan R; Connarn, Jamie; Ahmad, Atta; Young, Zapporah T; Kabza, Adam M; Zuiderweg, Erik R P; Sun, Duxin; Gestwicki, Jason E


    The rhodacyanine, MKT-077, has anti-proliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogs designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 ± 0.03 μM and 0.7 ± 0.2 μM, respectively). The analogs modestly destabilized the chaperone "clients", Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 MKT-077. These studies advance MKT-077 analogs as chemical probes for studying Hsp70's roles in cancer.

  5. An ELISA using recombinant TmHSP70 for the diagnosis of Taenia multiceps infections in goats. (United States)

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


    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.

  6. Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. (United States)

    Martinez, J; Perez-Serrano, J; Bernadina, W E; Rodriguez-Caabeiro, F


    Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology93, 171-180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with anti-hsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV + ALB produced less damage as manifested by fewer changes in the aforementioned damage parameters but PZ and PZ + ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression.

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

    DEFF Research Database (Denmark)

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


    The ability to express heat-shock proteins (HSP) under thermal stress is an essential mechanism for ectotherms to cope with unfavourable conditions. In this study, we investigate if Copper butterflies originating from different altitudes and/or being exposed to different rearing and induction...... temperatures show differences in HSP70 expression. HSP70 expression increased substantially at the higher rearing temperature in low-altitude butterflies, which might represent an adaptation to occasionally occurring heat spells. On the other hand, high-altitude butterflies showed much less plasticity...

  8. The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70. (United States)

    Roufayel, R; Johnston, D S; Mosser, D D


    Protein-damaging stress stimulates cell destruction through apoptosis; however, non-lethal proteotoxic stress induces an adaptive response leading to the increased synthesis of heat shock proteins, which inhibit apoptosis. In this study, we sought to determine the mechanism responsible for the accumulation of the BH3-only protein NOXA in heat-stressed cells and its prevention by the heat shock protein HSP70. Analysis of transcript levels by RT-qPCR revealed that miR-23a levels decreased in heat-stressed cells and that this was correlated with an increased abundance of NOXA mRNA, which contains a miR-23a binding site in its 3' untranslated region. Cells overexpressing HSP70 had higher levels of miR-23a, maintained these levels after heat shock and accumulated lower levels of NOXA mRNA and protein. The enhanced abundance of mir-23a in these HSP70-expressing cells is primarily due to its increased stability although higher levels of pri/pre-miR-23a expression, nuclear export and maturation were also contributing factors. Stable overexpression of miR-23a in the acute lymphoblastic T-cell line PEER resulted in reduced basal and heat-induced levels of NOXA mRNA and significantly inhibited heat-induced apoptosis. Additionally, stable overexpression of an shRNA targeting miR-23a in U937 lymphoma cells produced stable knockdown of miR-23a and resulted in increased NOXA mRNA and an increased sensitivity to heat-induced apoptosis. These results demonstrate the novel finding that hyperthermia affects the abundance of a microRNA that targets the expression of a pro-apoptotic protein and that HSP70 protects cells from heat-induced apoptosis by regulating the abundance of this microRNA. We speculate that the inhibition of miRNA transcription in heat-stressed cells could represent a general mechanism for apoptosis induction that is regulated by the molecular chaperone protein HSP70. Furthermore, we propose that HSP70 could be beneficial to tumor cells by helping to maintain the

  9. Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels. (United States)

    Miyata, Yoshinari; Li, Xiaokai; Lee, Hsiu-Fang; Jinwal, Umesh K; Srinivasan, Sharan R; Seguin, Sandlin P; Young, Zapporah T; Brodsky, Jeffrey L; Dickey, Chad A; Sun, Duxin; Gestwicki, Jason E


    The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

  10. Reduced Hsp70 and Glutamine in Pediatric Severe Malaria Anemia

    DEFF Research Database (Denmark)

    Kempaiah, Prakasha; Dokladny, Karol; Karim, Zachary


    Severe malarial anemia [SMA, hemoglobin (Hb) <5.0 g/dL] is a leading cause of global morbidity and mortality among children residing in Plasmodium falciparum transmission regions. Exploration of molecular pathways through global gene expression profiling revealed that SMA was characterized by dec...

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

    DEFF Research Database (Denmark)

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


    inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM...

  12. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

    DEFF Research Database (Denmark)

    Schmitz-Abe, Klaus; Ciesielski, Szymon J; Schmidt, Paul J


    The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases, characterized by defects in mitochondrial heme synthesis, iron-sulfur cluster (Fe-S) biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homologue located in the 5q...

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

    Directory of Open Access Journals (Sweden)

    Fresno Manuel


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

  14. The effect of glutamine infusion on the inflammatory response and HSP70 during human experimental endotoxaemia

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Pedersen-Skovsgaard, Theis; Mortensen, Ole Hartvig;


    INTRODUCTION: Glutamine supplementation has beneficial effects on morbidity and mortality in critically ill patients, possibly in part through an attenuation of the proinflammatory cytokine response and a stimulation of heat shock protein (HSP)70. We infused either alanine-glutamine or saline dur...

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

    Directory of Open Access Journals (Sweden)

    Renata Hernandes


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

  16. Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

    Energy Technology Data Exchange (ETDEWEB)

    Worrall, Liam; Walkinshaw, Malcolm D., E-mail: [School of Biological Sciences, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom)


    Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

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

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    Fedrigo Carlos A


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

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

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    Jian Ye

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

  19. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA. (United States)

    Rubtsova, Maria P; Sizova, Daria V; Dmitriev, Sergei E; Ivanov, Dmitri S; Prassolov, Vladimir S; Shatsky, Ivan N


    A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

  20. Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9. (United States)

    Nazir, Aamir; Mukhopadhyay, Indranil; Saxena, D K; Siddiqui, M Saeed; Chowdhuri, D Kar


    The study investigated the working hypothesis that a widely used fungicide captan exerts toxic effects on nontarget organisms. Transgenic Drosophila melanogaster (hsp70-lacZ) was used as a model by assaying stress gene expression as an endpoint for cytotoxicity and also to evaluate whether stress gene expression is sufficient enough to protect and to prevent tissue damage against toxic insult of the chemical. The study was further extended to understand the effect of the pesticide on development, life cycle, and reproduction of the organism and finally to evaluate a concentration of the chemical to be nontoxic to the organism. The study showed that (i) captan causes cytotoxicity at and above 0.015 ppm; (ii) at 0.0015 ppm captan, absence of hsp70 expression in the exposed organism was evaluated as the concentration referred to as no observed adverse effect level (NOAEL) for Drosophila; (iii) emergence pattern of flies was affected only at the highest concentration of captan by 4 days, while hatching and survivorship were unaffected even at this concentration; (iv) reproductive performance was significantly affected only at 125.0 and 1250.0 ppm captan, while in the lower dietary concentrations no such deleterious effects were observed; (v) at 1250.0 ppm, hsp70 failed to protect the cells from toxicant assault after 48 h exposure, thus leading to tissue damage as revealed by Trypan Blue staining. The present study shows the cytotoxic potential of captan and further reveals the application of stress genes in determining NOAEL and its expression as bioindicator of exposure to environmental contaminants.

  1. Study on relationship between porcine HSP70 protein expression and weaning diarrhoea piglets%仔猪HSP70蛋白表达与断奶腹泻关系研究

    Institute of Scientific and Technical Information of China (English)

    王莉; 汪蕊; 王希彪


    为了探讨HSP70在断奶过程中的作用,试验通过免疫组化方法检测民猪、长白猪断奶仔猪的胃、十二指肠、空肠、回肠、盲肠、结肠六个组织中HSP70蛋白的组织定位与定量变化.结果表明,胃肠道中HSP70蛋白主要在黏膜上皮和肠腺内表达,在各组织内的表达没有因腹泻与否而发生组织定位的改变,在除胃以外的其他消化道组织中,长白健康组仔猪的HSP70蛋白的表达量显著高于长白腹泻组、民猪健康与腹泻组.%In order to investigate the effect of HSP70 during weaning of piglets, this experiment determined the expression of HSP70 protein and located HSP70 protein in the gastrointestinal (Gl) tissues (stomach, duodenum, jejunum, ileum, cecum and colon) of Min pig and Landrace by immunohistochemistry. The results showed that, HSP70 protein expressed mainly in the epithelium mucosae and intestinal gland of Gl tract, while the diarrhoea didn't affect HSP70's location. The HSP70 expression of Landrace healthy piglets in Gl tract, expect for the stomach, was significantly higher than other groups (Landrace diarrhoea piglets, Min pig healthy and diarrhoea piglets).

  2. Characterization of a wheat HSP70 gene and its expression in response to stripe rust infection and abiotic stresses

    NARCIS (Netherlands)

    Duan, Y.H.; Guo, J.; Ding, K.; Wang, S.J.; Zhang, H.; Dai, X.W.; Chen, Y.Y.; Govers, F.; Huang, L.L.; Kang, Z.S.


    Members of the family of 70-kD heat shock proteins (HSP70 s) play various stress-protective roles in plants. In this study, a wheat HSP70 gene was isolated from a suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis f. sp. tritici. The gene, that

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

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    Bettencourt Brian R


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


    Directory of Open Access Journals (Sweden)

    N Shit


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

  5. Study of the role of Hsp70.1 in oridonin-treated hepatocarcinoma cells%Hsp70.1在冬凌草甲素处理的肝癌细胞中的作用研究

    Institute of Scientific and Technical Information of China (English)

    王辉; 叶燕; 黄树林; 禹志领


    目的 研究Hsp70.1在冬凌草甲素处理后的肝癌细胞HepG2中的表达情况及其与冬凌草甲素抗肿瘤活性的关系.方法 采用蛋白印迹法及实时荧光定量PCR技术观察Hsp70.1在冬凌草甲素处理的肝癌细胞HepG2的表达变化,并采用RNA干扰技术观察抑制Hsp70.1表达后对冬凌草甲素抗肝癌效果的影响.结果 冬凌草甲素作用12 h后,HepG2细胞的Hsp70.1蛋白及RNA表达均增加.Hsp70.1短发夹RNA(shRNA)转染抑制Hsp70.1表达后,与对照组相比,Hsp70.1 shRNA转染组、冬凌草甲素处理组或两者联合应用组的HepG2细胞的存活率均明显降低.结论 Hsp70.1可促进HepG2细胞的生存,对HepG2细胞和冬凌草甲素处理的HepG2细胞具有保护作用.%Objective To explore the expression of Hsp70. 1 in oridonin-treated hepatocarcinoma HepC2 cells and the correlation of Hsp70. 1 with the anticancer activity of oridonin. Methods By using Western blot and real-time PCR analysis,we tested the expression of Hsp70. 1 protein and mRNA in HepG2 cells treated with oridonin. After HepG2 cells were transfected with Hsp70.1 shRNA.the influence of Hsp70.1 on the anticancer activity of oridonin was investigated. Results The levels of Hsp70. 1 protein and mRNA increased in oridonin-treated HepG2 cells. Transient transfection of Hsp70. 1-specific shRNA suppressed Hsp70. 1 expression. Compared with control cells,Hsp70.1 shRNA-transfection,oridonin-treatment and combination of both-treatment groups significantly reduced cell viability. Conclusion Hsp70. 1 upregulation contributes to HepG2 cells survival,which suggests that Hsp70. 1 acts as a protective factor in both HepG2 cells and oridonin-treated HepG2 cells.

  6. Inhibition of hsp70 by methylene blue affects signaling protein function and ubiquitination and modulates polyglutamine protein degradation. (United States)

    Wang, Adrienne M; Morishima, Yoshihiro; Clapp, Kelly M; Peng, Hwei-Ming; Pratt, William B; Gestwicki, Jason E; Osawa, Yoichi; Lieberman, Andrew P


    The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

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

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    Elisa Zorzi


    Full Text Available 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.

  8. Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated. (United States)

    Dhankher, O P; Drew, J E; Gatehouse, J A


    A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5'-flanking sequence contains heat shock elements and other potential regulatory sequences.

  9. Nuclear HSP90 and HSP70 in COPD patients treated with formoterol or formoterol and corticosteroids

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    Holownia A


    Full Text Available Abstract Objective Heat shock proteins assist cellular protein folding and are required for the normal activity of steroid receptors. In this study we assessed nuclear HSP90 and HSP70 proteins and mRNA levels in cells isolated from induced sputum of chronic obstructive pulmonary disease patients treated for 4 weeks with formoterol (F or formoterol+budesonide (F/ICS. Methods Nuclear heat shock protein levels were assessed by Western blot and specific mRNAs were quantified in cell lysates using qRT-PCR. Results Both HSP90 and HSP70 protein levels were higher in the F/ICS-treated patients in comparison with the F-treated group (by 31%, P Conclusions It is possible that increased nuclear heat shock proteins may play a role in the attenuation of the response to glucocorticoids in COPD patients.

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

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


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

  11. Expression of class Ⅰ MHC molecule, HSP70 and TAP in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ling Deng; Wei Chen; Mei-Ying Cai; Da-Peng Wei


    AIM: To demonstrate whether class Ⅰ MHC molecule,transporter associated with antigen processing (TAP), and heat-shock proteion70 (HSP70) expressed in liver cancer cells before the design and construction of CTL vaccine against hepatocellular carcinoma (HCC).METHODS: We studied 30 HCC specimens by labeled streptavidin biotin (LSAB) method of immunohistochemistry.RESULTS: The results showed that the majority of HCC cells investigated naturally expressed class Ⅰ MHC and TAP,which were different from other tumor cells. Furthermore,we found that HSP70 expressed not only in cellular cytoplasm, but also on the cell surface in HCC.CONCLUSION: Our findings indicate that our understanding about immune escape mechanisms employed by HCC cells may be further improved. It is important to design and construct CTL vaccine against HCC.

  12. 旋毛虫HSP70基因的原核表达及其免疫原性%Prokaryotic expression of HSP70 gene of Trichinella spiralis and immunogenicity analysis of the expression product

    Institute of Scientific and Technical Information of China (English)

    徐佳; 禹洋; 唐颖; 俞昭旸; 李晓云; 宋铭忻


    利用RT-PCR方法扩增旋毛虫HSP70基因,将其扩增产物克隆到pEASY-Blunt Simple载体中,经限制性酶切后与pET-28a(+)载体连接,进一步转化至感受态细胞Transetta(DE3)中,用IPTG诱导表达重组旋毛虫HSP70。以纯化的重组HSP70制备多克隆抗体,并对HSP70进行免疫印迹分析及免疫组织化学染色。结果表明,重组质粒的鉴定和DNA测序均正确;SDS-PAGE和Western-blot分析结果显示,在70ku处获得一目的条带;免疫组织化学染色结果显示,该HSP70具有很好的免疫原性。证实,成功构建了重组表达质粒pET28a-HSP70,为深入研究旋毛虫HSP70的功能奠定了基础。%HSP70 gene of Trichinella spiralis was amplified by RT-PCR and then cloned into pEASY-Blunt Simple vector.The recombinant was digested and linked with pET-28a(+) vector.The recombinant plasmid was confirmed by PCR and DNA sequencing.The positive plasmid was transformed into competent Escherichia coli Transetta(DE3) and induced by IPTG for expression.The recombinant Trichinella HSP70 was purified to prepare polyclonal antibody.Trichinella HSP70 was analysed by Western-blot and stained by immunohistochemistry.The recombinant protein was confirmed to be about 70 ku in size.The positive signals were localized in the Trichinella by immunohistochemistry.The results showed that the recombinant expression plasmid pET28a-HSP70 was successfully constructed and laid the foundation for further studies on the function of HSP70.

  13. Transgenerational stress memory in Arabidopsis thaliana (L.) Heynh.: antioxidative enzymes and HSP70


    Ćuk, Katarina; Gogalo, Marko; Tkalec, Mirta; Vidaković-Cifrek, Željka


    Transgenerational transmission of information about stress exposure is manifested as an increase in the somatic homologous recombination frequency in plants. Our aim was to investigate whether information about changes of antioxidative enzyme activities and protein HSP70 induction are also transmitted in response to stress caused by UV-C irradiation. These stress indicators were investigated in Arabidopsis thaliana plants exposed to UV-C irradiation (6 and 600 J m-2) and its non-irradiated pr...

  14. Production and cleavage of Drosophila hsp70 transcripts extending beyond the polyadenylation site.


    Berger, S L; Meselson, M


    Transcription downstream of the polyadenylation site was studied in the Drosophila hsp70 gene, whose high level of transcription in response to temperature elevation facilitates detection of rare and possibly short-lived transcripts. Transcription downstream of the polyadenylation site was detected both in cultured cells and in intact animals. Even shortly after temperature elevation the extended nonpolyadenylated RNAs were rare relative to mature message, and their level continued to increas...

  15. Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

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    Nadezda Kust

    Full Text Available The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

  16. Isoforms of Hsp70-binding human LDL in adult Schistosoma mansoni worms. (United States)

    Pereira, Adriana S A; Cavalcanti, Marília G S; Zingali, Russolina B; Lima-Filho, José L; Chaves, Maria E C


    Schistosoma mansoni is one of the most common parasites infecting humans. They are well adapted to the host, and this parasite's longevity is a consequence of effective escape from the host immune system. In the blood circulation, lipoproteins not only help to conceal the worm from attack by host antibodies but also act as a source of lipids for S. mansoni. Previous SEM studies showed that the low-density lipoprotein (LDL) particles present on the surface of adult S. mansoni worms decreased in size when the incubation time increased. In this study, immunocytochemical and proteomic analyses were used to locate and identify S. mansoni binding proteins to human plasma LDL. Ultrathin sections of adult worms were cut transversely from the anterior, medial and posterior regions of the parasite. Immunocytochemical experiments revealed particles of gold in the tegument, muscle region and spine in male worms and around vitelline cells in females. Immunoblotting and 2D-electrophoresis using incubations with human serum, anti-LDL antibodies and anti-chicken IgG peroxidase conjugate were performed to identify LDL-binding proteins in S. mansoni. Analysis of the binding proteins using LC-MS identified two isoforms of the Hsp70 chaperone in S. mansoni. Hsp70 is involved in the interaction with apoB in the cytoplasm and its transport to the endoplasmic reticulum. However, further studies are needed to clarify the functional role of Hsp70 in S. mansoni, mainly related to the interaction with human LDL.

  17. Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease. (United States)

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


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

  18. Polyglutamine expansion in Drosophila: thermal stress and Hsp70 as selective agents

    Indian Academy of Sciences (India)

    Brian R Bettencourt; Catherine C Hogan; Mario Nimali


    Repetitive DNA sequences that encode polyglutamine tracts are prone to expansion and cause highly deleterious phenotypes of neurodegeneration. Despite this tendency, polyglutamine tracts (``polyQs”) are conserved features of eukaryotic genomes. PolyQs are the most frequent protein-coding homotypic repeat in insect genomes, and are found predominantly in genes encoding transcription factors conserved from Drosophila through human. Although highly conserved across species, polyQ lengths vary widely within species. In D. melanogaster, polyQs in 25 genes have more alleles and higher heterozygosity than all other poly-amino acid tracts. The heat shock protein Hsp70 is a principal suppressor of polyQ expansions and may play a key role in modulating the phenotypes of the alleles that encode them. Hsp70 also promotes tolerance of natural thermal stress in Drosophila and diverse organisms, a role which may deplete the chaperone from buffering against polyQ toxicity. Thus in stressful environments, natural selection against long polyQ alleles more prone to expansion and deleterious phenotypes may be more effective. This hypothesis can be tested by measuring the phenotypic interactions between Hsp70 and polyQ transgenes in D. melanogaster undergoing natural thermal stress, an approach which integrates comparative genomics with experimental and ecological genetics.

  19. HSF1-TPR interaction facilitates export of stress-induced HSP70 mRNA. (United States)

    Skaggs, Hollie S; Xing, Hongyan; Wilkerson, Donald C; Murphy, Lynea A; Hong, Yiling; Mayhew, Christopher N; Sarge, Kevin D


    Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.

  20. Hsp70 confines tumor progression of rat histiocytoma and impedes the cytotoxicity induced by natural killer cells and peritoneal macrophages

    Institute of Scientific and Technical Information of China (English)

    Amere Subbarao Sreedhar


    Objective:To study the role of inducible form of heat shock protein 70 (Hsp70) in the host tumor regression of rat tumor model.Methods: We examined the role of Hsp70 in host tumorigenicity andin vitro cellular cytotoxicity using a rat histocytoma. The differential tumor growth and regression kinetics were studied and correlated with the expression of Hsp70, activation of macrophages and natural killer (NK) cells, and circulating or tumor infiltrating immune molecules in the host system.Results: The sub cuteaneous (s.c.) tumor regression was correlated with increased serum cytokines such as IL-12, TNFα,IFNγ and Hsp70. Despite of similar increase of Hsp70 in intraperitoneal (i.p.) tumor implanted animals, animals succumb to tumor growth, further, evidently, no immune molecule activation was observed. The viral promoter driven Hsp70 over expression in these tumor cells restrained solid tumor growth, however, failed to inhibit ascites growth. The NK cells from s.c. immunized animals induces cytotoxicity in the presence of anti-tumor antibody, which necessitated CD40-L expression, conversely, NK cells from i.p. immunized animals failed to induce cytotoxicity. The NK cells from s.c. or i.p. implanted animals with Hsp70 positive tumor cells failed to induce such cytotoxicity. The peritoneal macrophages isolated from s.c. tumor implanted animals when co-cultured with parental BC-8 cells lyses tumor cells, nevertheless entail macrophage specific TNFα expression. On the contrary, Hsp70 expressing BC-8 tumor cells were resistant to peritoneal macrophage induced cytolysis.Conclusions:This study brings out that Hsp70 possibly involved in regulating the host tumor response and cellular cytotoxicity.

  1. 草鱼hsp70和hsp90对温度急性变化的响应%Effect of rapid temperature change on expression of hsp70 and hsp90 in grass carp(Ctenopharyngodon idella)

    Institute of Scientific and Technical Information of China (English)

    周鑫; 董云伟; 王芳; 董双林


    Grass carp ( Ctenopharyngodon idella) is one of the most important cultured fishes in China. During transportation in summer,massive mortality often occurs. As molecular chaperones,Hsps assist cells in their recovery from stress and promote cytoprotection. Limited reports described the expression of Hsps in grass carp at high temperatures, and the relationship between oxygen consumption, expression of heat shock protein and mortality needs to be investigated based on the oxygen- and capability- limited thermal tolerance theory (OCLTT) to further elucidate the thermal adaptation of this species to high temperature in view of integrated biology. In the present study,the expressions of hsp70 and hsp90 after rapid temperature increase were studied. Grass carps were acclimated at 20 t and then exposed to designated temperatures(20,22,24, 26,28,30,32,34 ℃) for 3 h heat shock. After 2 h-recovery at 20℃, liver, muscle and gill of tested fish were sampled and hsp70 and hsp90 were determined using semi-quantitive real-time PCR. Expression of hsps increased with temperature increase until 32℃ in muscle and gill, and then decreased at 34 ℃. In liver, expression of hsps kept increasing in all temperature treatments. These results indicated that hsp70 and hsp90 were sensitive to temperature increase in grass carp. Analyzed heat shock proteins data with previous results of oxygen consumption and lethal temperatures of grass carp, the physiological adaptations of grass carp could be explained by oxygen- and capability- limited thermal tolerance theory (OCLTT), which pointed out that the imbalance between oxygen demand and tissue oxygen supply ability was the primary factor which limited organisms' thermal tolerance and the ability of aerobic metabolism greatly limited the survival of organism beyond critical temperatures. With temperature increase, the maximum value of oxygen consumption in grass carp occurred at 28 ℃, and then the oxygen consumption decreased when

  2. An inducible HSP70 gene from the midge Chironomus dilutus: Characterization and transcription profile under environmental stress (United States)

    Karouna-Renier, N. K.; Rao, K.R.


    In the present study, we identified and characterized an inducible heat shock protein 70 (HSP70) from the midge Chironomus dilutus and investigated the transcriptional profile of the gene under baseline and environmentally stressful conditions. Using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we observed increased expression of CD-HSP70-1 in response to both heat shock and copper stress. We also investigated the expression of this gene during midge development. All C. dilutus developmental stages expressed CD-HSP70-1 under normal conditions, although at extremely low levels. Phylogenetic analysis of the amino acid sequence demonstrated distinct clustering of this gene with inducible HSP70s from other insect species. ?? 2008 The Authors.

  3. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy


    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing


    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The effica...

  4. Elimination of Enhanced Thermal Resistance of Spheroid Culture Model of Prostate Carcinoma Cell Line by Inhibitors of Hsp70 Induction

    Directory of Open Access Journals (Sweden)

    Samideh Khoei


    Full Text Available AbstractObjective: The purpose of this study was to investigate the enhanced thermal resistancemechanism of the DU145 tumor spheroid cultures as compared to the prostate carcinomacell line's monolayer cultures.Materials and Methods: DU145 cells were cultured either as spheroids or monolayers.Cultures were treated with hyperthermia in a precision water bath (at 43°C for 60 minutesand/or quercetin (50 and 500 μM for monolayer and spheroid cultures respectively. Afterhyperthermic treatment, the cell viability colony forming ability, and the expression of heatshock protein 70 (Hsp70 were examined in both culture systems. Hsp70 expression wasstudied using the western blot method.Results: Our results showed that the DU145 monolayer and spheroid cell culture treatmentwith hyperthermia alone resulted in a marked survival inhibition. Furthermore, thespheroids showed a more significant resistance to hyperthermia compared to the monolayercultures (p = 0.01. They also produced more Hsp70 than the monolayer cultures.Treatment of cells with quercetin reduced the Hsp70 level in both culture systems. However,with the reduced Hsp70 levels, thermal resistance of the spheroids showed a greaterdecrease in relation to that of the monolayers.Conclusion: The results suggest that the enhanced hyperthermia resistance mechanismof the spheroid cultures compared to that of the monolayer cultures can be attributed tospheroids' Hsp70 production.

  5. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy. (United States)

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing


    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.

  6. Involvement of serum HSP 70 in Guillain-Barré Syndrome: An exploratory study and a review of current literature

    Directory of Open Access Journals (Sweden)

    Aida Loshaj-Shala


    Full Text Available The evolutionary conserved family of heat shock proteins (HSP is responsible for protecting cells against different types of stress. Although the levels of HSP can be readily measured in serum, the levels of HSP 70 in patients Guillain-Barre Syndrome (GBS have not been studied before. To this aim we investigate whether patients with GBS (n=21 had altered serum HSP 70 levels compared to healthy controls (HC, n=9 and to patients affected by other immune disorders such as multifocal motor neuropathy (MMN, n=4 and chronic inflammatory demyelinating polyneuropathy (CIDP, n=6. The highest HSP 70 value (15.78 ± 1.72 ng/mL was found in one patient in the GBS group, although we have found that serum HSP70 levels were significantly higher in 2 out of the 21 GBS patients (9.5%. Hence, it is of interest to underline that the patient with the highest HSP70 level, had also the best recovery rate. Моrе extensive research is required in order to support the hypothesis that HSP 70 serum concentration may be a useful biomarker for the prediction of remission outcome for GBS patients.

  7. Effect of whey proteins in the system defense HSP 70 and biochemical parameters in rats


    Carolina Soares de Moura


    Resumo: As heat shock proteins (HSPs), ou proteínas do estresse, correspondem a um importante sistema de defesa celular que é capaz de proteger e reparar danos causados ao organismo, conferindo à célula maior tolerância e resistência contra situações de alteração na homeostase, sendo também consideradas como um sistema antioxidante complementar. A glutamina é conhecida pelo seu potencial em promover o aumento na HSP70 contra diversas situações agressoras. As proteínas do soro do leite (PSL) c...

  8. Positive and negative regulation of basal expression of a yeast HSP70 gene.


    Park, H O; Craig, E A


    The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-low...

  9. Characterizing HSF1 Binding and Post-Translational Modifications of hsp70 Promoter in Cultured Cortical Neurons: Implications in the Heat-Shock Response.

    Directory of Open Access Journals (Sweden)

    Andrea V Gómez

    Full Text Available Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1 and histone posttranslational modifications (PTMs at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression.

  10. Chromosomal localization of heat shock protein 70 (HSP70) gene in Zhikong scallop Chlamys farreri using BAC-FISH%栉孔扇贝热休克蛋白70(HSP70)基因的BAC-FISH定位

    Institute of Scientific and Technical Information of China (English)

    郇聘; 张晓军; 李富花; 张洋; 赵翠; 刘保忠; 相建海


    Zhikong scallop (Chlamys farreri) is one of key species in the marine aquaculture of China. Till present, the disease control is the main issue and many subjects were focused on disease- resistance and stress-related genes. Heat shock protein 70 (HSP70) shows diverse functions in the anti-stress reaction and pathogen-resistance reaction. However, the studies on HSP70 in C. farreri were mainly on a sequence analysis and expression profile, there is no result about the gene localization of HSP70. In this study, with the support of a BAC DNA library of C. farreri, a HSP70-eontaining BAC clone, CFB 123C08, was mapped to the long arms of a pair of homologous chromosomes of C. farreri using BAC-FISH. This study would facilitate further studies on the HSP70 of C. farreri. And as the first attempt of chromosomal mapping of low-copy genes in C. farreri, this study would promote the researches on the chromosome identification of C farreri.%利用BAC-FISH技术,将包含HSP70基因的BAC克隆定位到栉孔扇贝的一对同源染色体的长臂上.HSP70基因的染色体定位将对深入研究该基因的结构及功能并将其应用于生产实践提供基础支持.同时,本研究是首次对栉孔扇贝低拷贝基因进行染色体定位的实践,其结果将为栉孔扇贝的染色体的深入研究以及染色体鉴别等工作提供必要的参考.

  11. hsp70 genes in the human genome: Conservation and differentiation patterns predict a wide array of overlapping and specialized functions

    Directory of Open Access Journals (Sweden)

    Macario Alberto JL


    Full Text Available Abstract Background Hsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago; some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family. Results We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD was not. Nine proteins were typical Hsp70s (65–80 kDa with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages. Conclusion The human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into

  12. Isolation and Identification of Mycoplasma ovipneumoniae and Genetic Diversity of HSP70 Gene%绵羊肺炎支原体的分离鉴定及其 HSP70基因遗传多样性

    Institute of Scientific and Technical Information of China (English)

    胡政香; 乔军; 孟庆玲; 张金生; 陈创夫


    The colonial morphology,biochemical indexes and molecular biology of the pathogens isolated from lung samples of 7 suspected M.ovipneumoniae infection sheep farms were analyzed to discuss genetic diversity of M.ovipneumoniaeep epidemic strain in Xinjiang.Results:5 isolated strains all are M. ovipneumoniae.The colony is papillary and thallus is polymorphism.15 isolated strains can hydrolyze glucose,but can not hydrolyze arginine and urea.15 isolated strains can fade and adsorb methylene blue milk,which results in erythrocytes hemolysis.The size of 16Sr RNA sequence is 361 bp.The nucleotide homology and amino acid sequence homology of HSP70 gene among 5 isolated strains are 94.1% and 93.2%respectively.No.593 locus of the isolated strains is inserted with Gln compared with the standard strain Y98.There is genetic diversity in the isolated strains in Xinjiang.%为探明新疆地区绵羊肺炎支原体(Mycoplasma ovipneumoniae ,MO)流行株是否存在遗传多样性,用 KM2培养基对塔城地区7个羊场疑似 MO 感染的肺脏病料进行病原分离,并进行菌落形态、生化试验和分子生物学鉴定。结果表明:15株分离均为 MO,菌落呈乳头状,菌体呈多形性;能水解葡萄糖但不能水解精氨酸、尿素,可使美兰牛乳褪色、吸附并引起红细胞溶血;16Sr RNA 序列大小为361 bp。其中,5株 MO HSP 70基因的核苷酸同源性为94.1%,氨基酸同源性为93.2%,与 MO 标准毒株 Y98相比,分离株在593位点均插入 Gln(谷氨酰胺)。MO 新疆分离株已发生遗传变异,存在遗传多样性。

  13. Tumor tissue characterization evaluating the luciferase activity under the control of a hsp70 promoter and MR imaging in three tumor cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Hundt, Walter [Department of Radiology, Lucas MRS Research Center, Stanford School of Medicine, Stanford, CA 94305 (United States); Department of Clinical Radiology, University of Munich (Germany)], E-mail:; Steinbach, Silke [Department of Otolaryngology-Head and Neck Surgery, Technical University of Munich (Germany); O' Connell-Rodwell, Caitlin E. [Department of Pediatrics, Microbiology and Immunology and Radiology, Stanford School of Medicine, Stanford, CA 94305 (United States); Mayer, Dirk; Bednarski, Mark D.; Guccione, Samira [Department of Radiology, Lucas MRS Research Center, Stanford School of Medicine, Stanford, CA 94305 (United States)


    We investigated the luciferase activity under the control of a hsp70 promoter and MR imaging for three tumor cell lines. Three tumor cell lines, SCCVII, NIH3T3 and M21 were transfected with a plasmid containing the hsp70 promoter fragment and the luciferase reporter gene and grown in mice. Bioluminescence imaging of the tumors was performed every other day. MR imaging, pre- and post-contrast T1-wt SE, T2-wt FSE, Diffusion-wt STEAM-sequence, T2-time determination were obtained on a 1.5-T GE MRI scanner at a tumor size of 600-800 mm{sup 3} and 1400-1600 mm{sup 3}. Comparing the different tumor sizes the luciferase activity of the M21 tumors increased about 149.3%, for the NIH3T3 tumors about 47.4% and for the SCCVII tumors about 155.8%. Luciferase activity of the M21 tumors (r = 0.82, p < 0.01) and the SCCVII tumors (r = 0.62, p = 0.03) correlated significant with the diffusion coefficient. In the NIH3T3 tumors the best correlation between the luciferase activity and the MRI parameter was seen for the SNR (T2) values (r = 0.78, p < 0.01). The luciferase activity per mm{sup 3} tumor tissue correlated moderate with the contrast medium uptake (r = 0.55, p = 0.01) in the M21 tumors. In the NIH3T3 and SCCVII tumors a negative correlation (r = -0.78, p < 0.01, respectively, r = -0.49, p = 0.02) was found with the T2 time. Different tissue types have different luciferase activity under the control of the same hsp70 promoter. The combination of MR imaging with bioluminescence imaging improves the characterization of tumor tissue giving better information of this tissue on the molecular level.

  14. Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis. (United States)

    Batista, Fernanda A H; Seraphim, Thiago V; Santos, Clelton A; Gonzaga, Marisvanda R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C


    Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

  15. 赤拟谷盗HSP70基因克隆和竞争定量PCR检测%Cloning and quantitative competitive PCR assay of HSP70 gene in Tribolium castaneum ( Coleoptera: Tenebrionidae)

    Institute of Scientific and Technical Information of China (English)

    苏丽娟; 董晓慧; 尹新明


    To study the expression changes of heat shock protein 70 (a highly conserved protein) in Tribolium castaneum after exposure to heat stress, a fragment of 681 bp encoding for HSP70 was amplified and sequenced from T.castaneum.The fragment encoded 227 amino acid residues with the GenBank accession no.HM345948.The result of homology analysis showed that this fragment shared 97% identity with hsp70 from L.decemlineata (AF322911.1 ).Comparison of the deduced amino acid sequences of HSP70 in T.castaneum with those in Leptinotarsa decemlineata, Mamestra brassicae, Drosophila melanogaster and Liriomyza sativae indicated that they shared more than 94% identity.The internal competitor used in quantitative competitive PCR was obtained by RT-PCR.The detection system of hsp70 was constructed by PCR amplification using the same quantity of target cDNA and a series of diluted internal competitor as template.The linear equation of standard curve was Y= 1.032X- 1.618 (r2 =0.975).This study provides a very convenient method for the quantitation of the hsp70 expression changes in T.castaneum and offers the basic data for the prevention and control of pests using thermal control technology.%为研究赤拟谷盗Tribolium castaneum受到热胁迫后高度保守的热激蛋白70(heat shock protein 70,HSP70)基因的表达变化,本研究扩增了681 bp的赤拟谷盗hsp70片段,编码227个氨基酸残基,GenBank登录号为HM345948.同源性分析表明:赤拟谷盗hsp70核苷酸序列与马铃薯甲虫Leptinotarsa decemlineata的hsp70(GenBank登录号:AF322911.1)同源性最高,为97%;其推测的蛋白序列与马铃薯甲虫、甘蓝夜蛾Mamestra brassicae、黑腹果蝇Drosophila melanogaster和美洲斑潜蝇Liriomyza,sativae的HSP70蛋白均有94%以上的同源性.利用RT-PCR技术得到与赤拟谷盗hsp70进行竞争定量的内部竞争物,以等量的目标cDNA和一系列稀释的竞争模板进行竞争PCR扩增,构建了hsp70的竞争定量PCR检测体系,该

  16. The expression of HSP70 in dental pulp tissues in early orthodntic force and its clinical significance%矫治力初期牙髓组织 HSP70表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    赵紫婷; 苏士文; 蒋蓉妹


    目的:观察在矫治力初期牙髓组织中 heat shock protein 70(HSP70)的分布及表达,探讨适宜的矫治力对牙髓组织的影响并正确运用生物力学原则。方法16例采用拔除上颌双侧第一前磨牙进行固定矫治的患者,随机分为正常对照组,加力(0.5 N)1、3、7 d 组在受力相应时段拔除,并制备石蜡标本。免疫组织化学法(SABC)观察牙髓中 HSP70的表达及分布。结果HSP70的阳性反应产物呈深褐色均质性沉淀,主要在血管内皮细胞,成牙本质细胞胞质周围呈颗粒状阳性表达。在加力1 d组 HSP70的表达并没有明显增强(P >0.05),3天组表达达高峰尤其是在血管内皮细胞中(P <0.01),到7天后牙髓细胞的整体表达呈弱阳性。所有病例无一例牙髓坏死。结论在牙移动过程中牙髓组织中 HSP70的表达先升高后又逐渐减低至恢复正常水平,提示 HSP70在牙移动过程中对牙髓组织的改建过程可能起着重要作用;适宜的矫治力作用下产生的 HSP70可能对牙髓组织提供了有效的保护。%Objective To detect the expression and distribution of HSP70 in human dental pulp tissues and to analyze the influences of orthodontic forces on dental pulp and to use biomechanic principle correctly .Methods 16 patients were randomly assigned to con-trol group and 1 d,3d and 7d groups after orthodontics.The test teeth were extracted at the corresponding time and paraffin was pre-pared.Immunohistochemical method (SABC method)was utilized to localize and examine the expression of HSP70.Results HSP70 was stained inodontoblast,vascular enelothelial cells and some smooth muscle cells.The expression of HSP70 didn′tincreaseobviously in 1 d group (P >0.05);it reached the highest level in 3d group especially in smooth muscle cell of vessel wall(P <0.01 )and decreased greatly in 7d group.There was no pulp necrosisin all cases.Conclusions The changes of HSP70 in

  17. HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

    Institute of Scientific and Technical Information of China (English)

    ZHU Liqin; LI Hui; XIONG Jinhu; WANG Tongxiang; OU Xuan; WEI Yun; WU Xinxing


    Using human papillomavirus type 16 (HPV16) E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7C91G gene. Mice, after being immunized with E7C91G-HSP70, E7C91G/HSP70, E7C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8+ T-cell precursor frequencies oF280. 33±2.52, 144.34±4. 04, 164.34±5.13 and 82.33± 3.51 respectively within every 1 × 105 mouse splenocytes. This proves that E7C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body(p<0.01). After being immunized with E7C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all,E7C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than that of wild type E7 DNA vaccine.

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

    Directory of Open Access Journals (Sweden)

    Huei-Fen Jheng


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

  19. Quercetin对晶状体上皮细胞HSP70、HSP27表达的调节作用%HSP70 and HSP27 expression in lens epithelial cells modulated by Quercetin

    Institute of Scientific and Technical Information of China (English)

    饶惠英; 姚克; 汤霞靖; 徐雯


    目的研究大鼠眼钝挫伤后晶状体上皮细胞(LECs)热休克蛋白(HSP)70、HSP27的表达,并给予喂饲Quercetin(HSP阻滞剂),观察Quercetin对LECs HSP70及HSP27表达的调节.方法SD大鼠48只,随机分成拍打组和Quercetin组,每组各24只24眼,右眼为实验眼.拍打组:20 g铁球20 cm高度拍打眼球100次.Quercetin组:给大鼠喂饲Quercetin(100 mg/kg),2~3 h后再拍打眼球.RT-PCR检测LECs HSP70、HSP27基因表达.结果钝挫性眼外伤可造成LECs HSP70基因表达的增强,拍打眼球后1 h HSP70表达开始升高,3 h后达到高峰,24 h后降至正常.Quercetin组HSP70基因表达随时间亦出现相应的提高,但与拍打组相比其峰值下降,差异有非常显著性意义.两组HSP27基因表达均无明显改变.结论钝挫性眼外伤中LECsHSP70表达的增强提示HSP70可能在钝挫性外伤性白内障形成过程中对晶状体变性蛋白起保护作用,预先喂饲Quercetin可抑制LECsHSP70基因的表达,其作用机制可能发生于HSP转录水平.

  20. 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:; 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)


    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)

  1. Priming the immune system of Penaeid shrimp by bacterial HSP70 (DnaK). (United States)

    Phuoc, L H; Hu, B; Wille, M; Hien, N T; Phuong, V H; Tinh, N T N; Loc, N H; Sorgeloos, P; Bossier, P


    This study was conducted to test the effect of DnaK on priming immune responses in Penaeid shrimp. Juvenile-specific pathogen-free (SPF) P. vannamei shrimp were injected with 0.05 μg recombinant DnaK. One hour post-DnaK priming, a non-lethal dose of Vibrio campbellii (10(5) CFU shrimp(-1)) was injected. Other treatments include only DnaK or V. campbellii injection or control with blank inocula. The haemolymph of three shrimp from each treatment was collected at 1.5, 6, 9 and 12 h post-DnaK priming (hpp). It was verified that injection with DnaK and V. campbellii challenge affected the transcription of 3 immune genes, transglutaminase-1 (TGase-1), prophenoloxidase-2 (proPO-2) and endogenous HSP70 (lvHSP70). In P. monodon, shrimp were first injected with DnaK at a dose of 10 μg shrimp(-1) and one hour later with 10(6) CFU of V. harveyi (BB120) shrimp(-1). Shrimp injected with DnaK showed a significant increase in proPO expression compared to the control (P < 0.05). Yet a double injection (DnaK and Vibrio) seemed to cause an antagonistic response at the level of expression, which was not equalled at the level of PO activity. Those results suggest that DnaK is able to modulate immune responses in P. vannamei and P. monodon.

  2. Expression, Purification and Immunologic Identification of the Recombinant HSP70 Gene From Echinococcus Granulosus%细粒棘球蚴重组HSP70基因的表达、纯化及免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    于晶晶; 于辛酉; 王娅娜; 师志云; 马锐; 卜阳; 罗永云; 赵巍


    目的 构建细粒棘球蚴(Echinococcus granulosus,Eg)热休克蛋白70(Heat shock protein,HSP70)基因的重组质粒并原核表达、纯化及对其免疫特性进行鉴定.方法 从重组质粒HSP70/pGEM-T中酶切HSP70目的片段,亚克隆入表达载体pET-28a,转化入大肠杆菌E.coli BL21(DE3)plysS进行融合表达,经His-band树脂纯化试剂盒小量纯化,SDS-PAGE 和Western blot方法鉴定表达产物.结果 成功构建Eg.HSP70/ pET-28a/ E.coli BL21基因工程菌株,诱导表达重组蛋白和纯化分离得到14kDa Eg.HSP70均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.结论 初步证实该重组蛋白具有较好的抗原性.

  3. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  4. Effects of heat stimulation via microwave applicator on cartilage matrix gene and HSP70 expression in the rabbit knee joint. (United States)

    Tonomura, Hitoshi; Takahashi, Kenji A; Mazda, Osam; Arai, Yuji; Shin-Ya, Masaharu; Inoue, Atsuo; Honjo, Kuniaki; Hojo, Tatsuya; Imanishi, Jiro; Kubo, Toshikazu


    The objective of this study was to investigate the effects of heat stimulation on the expression of extracellular matrix genes and heat-shock protein 70 (HSP70) in rabbit articular cartilage in vivo. Heat stimulation was applied to the knee joints of Japanese white rabbits for 20 min using a microwave (MW) applicator (2.45-GHz, 0-80 W). After 8-72 h, the articular cartilage was removed from the knee joints and proteins and total RNA were extracted. As controls, knee joints without heat stimulation were analyzed. The expression of HSP70 was confirmed by real-time PCR and Western blotting. The expression of proteoglycan core protein (PG) and type II collagen (Col II) was quantified using real-time PCR to assess cartilage matrix metabolism. Compared to controls, HSP70 expression was higher with more than 40 W of heat stimulation. The expression of PG and Col II mRNA was higher, with more than 20 W of heat stimulation and peaked with 40 W. When quercetin was used to inhibit the induction of HSP70 expression, PG mRNA expression did not increase. External MW application stimulated HSP70 expression in the articular cartilage in vivo. The expression of extracellular matrix genes was increased by appropriate heat stimulation.

  5. 肉鸡热应激下肝脏和下丘脑HSP70mRNA的表达%Expression of HSP70 mRNA in liver and hypothalamus in chicken under heat stress

    Institute of Scientific and Technical Information of China (English)

    许生友; 陈兴勇; 姜润深; 耿照玉


    研究优质黄羽肉鸡热应激期间肝脏和下丘脑组织中热休克蛋白70(heat shock protein 70,HSP70)基因mRNA的动态表达规律,为揭示该类型优质鸡种热应激反应机理提供参考.64日龄Anak 40(♂)×岭南黄(♀)杂交后代母鸡置于(38±1)℃恒温室内进行持续热应激处理,采取荧光定量PCR方法分别测定热应激起始(0 h)、热应激后12、24、36、48和60 h肝脏和下丘脑组织中HSP70 mRNA的表达水平.随着热应激时间的延长,肝脏和下丘脑组织中HSP70 mRNA表达水平均显著上升,至热应激36 h时表达水平达高峰值,约为应激起始表达水平的7倍,差异显著(P<0.05);热应激48和60 h,下丘脑HSP70 mRNA表达量快速回落至热应激初期的水平,而肝脏表达量极显著地骤降至低于热应激初期10%的水平.在(38±1)℃持续热应激期间,优质肉鸡肝脏和下丘脑HSP70 mRNA表达呈现基本同步的变化规律,即表达水平先上升后下降,热应激36 h时表达水平达高峰值.%This study is to investigate the dynamic expression profiles of HSP70 mRNA in liver and hypothalamus in quality chicken under heat stress,and explore the mechanism of heat stress reaction of this kind of chicken. The 64-day- old subjects produced from Anak 40 ( ♂ ) × Lingnan Yellow ( ♀ ) were placed in a room with the ambient temperature at (38 ± 1)℃ for heat stress treatment. The HSP70 mRNA levels in liver and hypothalamus were detected by fluorescence quantitative PCR at the initial, 12,24,36,48,and 60 h of the stress,respectively. During heating stress,the expression of HSP70 mRNA in the liver and hypothalamus markedly increased, and reached the peak at 36 h, which significantly (P<0.05) higher than the initial level by approximately 7-fold. At 48 and 60 h of the stress, the HSP70 mRNA in hypothalamus decreased dramatically to the level as at the initial of heat stress. While in liver, the HSP70 mRNA significantly decreased to the level less than 10

  6. Erianthus arundinaceus HSP70 (EaHSP70) Acts as a Key Regulator in the Formation of Anisotropic Interdigitation in Sugarcane (Saccharum spp. hybrid) in Response to Drought Stress. (United States)

    Augustine, Sruthy Maria; Cherian, Anoop V; Syamaladevi, Divya P; Subramonian, N


    Plant growth during abiotic stress is a long sought-after trait especially in crop plants in the context of global warming and climate change. Previous studies on leaf epidermal cells have revealed that during normal growth and development, adjacent cells interdigitate anisotropically to form cell morphological patterns known as interlocking marginal lobes (IMLs), involving the cell wall-cell membrane-cortical actin continuum. IMLs are growth-associated cell morphological changes in which auxin-binding protein (ABP), Rho GTPases and actin are known to play important roles. In the present study, we investigated the formation of IMLs under drought stress and found that Erianthus arundinaceus, a drought-tolerant wild relative of sugarcane, develops such growth-related cell morphological patterns under drought stress. Using confocal microscopy, we showed an increasing trend in cortical F-actin intensity in drought-tolerant plants with increasing soil moisture stress. In order to check the role of drought tolerance-related genes in IML formation under soil moisture stress, we adopted a structural data mining strategy and identified indirect connections between the ABPs and heat shock proteins (HSPs). Initial experimental evidence for this connection comes from the high transcript levels of HSP70 observed in drought-stressed Erianthus, which developed anisotropic interdigitation, i.e. IMLs. Subsequently, by overexpressing the E. arundinaceus HSP70 gene (EaHSP70) in sugarcane (Saccharum spp. hybrid), we confirm the role of HSP70 in the formation of anisotropic interdigitation under drought stress. Taken together, our results suggest that EaHSP70 acts as a key regulator in the formation of anisotropic interdigitation in drought-tolerant plants (Erianthus and HSP70 transgenic sugarcane) under moisture stress in an actin-mediated pathway. The possible biological significance of the formation of drought-associated interlocking marginal lobes (DaIMLs) in sugarcane plants upon

  7. 猪带绦虫HSP70-4的真核表达及其抗血清的制备%Eukaryotic expression and anti-serum preparation of HSP70-4 from Taenia solium

    Institute of Scientific and Technical Information of China (English)

    殷静; 王帅; 刘光学; 何伟; 才学鹏


    为了研究猪带绦虫(Taenia solium)热休克蛋白70-4 (TsHSP70-4)基因序列的特征及其真核表达蛋白的抗原性,参考GeneDB中猪带绦虫基因组注释信息,设计特异性引物,RT-PCR扩增TsHSP70-4ORF序列.根据毕赤酵母密码子偏好性,对TsHSP70-4基因密码子进行优化,连接载体pPIC9K,在毕赤酵母中进行诱导表达,通过Western-blot及质谱测序进行蛋白鉴定.将纯化后的TsHSP70-4表达蛋白免疫新西兰大白兔,制备抗血清.结果显示,成功克隆出大小为1 953 bp的TsHSP70-4ORF序列.在毕赤酵母中进行了高效表达,获得大小约为95 ku的重组蛋白.Western-blot检测和质谱鉴定表明,获得的重组蛋白即为TsHSP70-4.免疫新西兰大白兔,制备出效价高达1∶409 600的抗血清.该研究为猪囊尾蚴病新疫苗的研制提供了依据.

  8. HSP70 expression of workers exposure to power frequency electromagnetic fields in 500 kV substation. 'WEN%500kV变电站工人HSP70表达水平研究

    Institute of Scientific and Technical Information of China (English)

    温翠菊; 闫雪华; 李丽; 黎丽春; 张丹英; 邹剑明


    Objective To explore the HSP70 expression of workers exposure to power frequency electromagnetic fields in 500 kV substation. Methods 263 workers and 252 administrative staff were selected as exposed and control group, respectively. The contents of HSP70 protein in sera were determined by ELISA assay. Results The differcnce of power frequency electric field and power frequency magnetic field between exposed and control group was statistically significant (P=0.000), (P=0.000), respectively. HSP70 protein contents were (17.71±13.63) and (16.65±9.60), respectively. The difference was not statistically significant (P=0.430). Conclusion The contents of HSP70 protein of workers exposure to power frequency electromagnetic fields in 500 kV substation are no different from the control group.%目的 探讨工频电磁场对500 kV变电站工人外周血HSP70表达水平的影响,寻找可实际应用早期效应标记物.方法 选择广东省内500kV变电站263名巡检人员作为暴露人群及抽取252名各变电站行政管理人员作为对照人群,分别抽取外周血5 ml,用HSP70 ELISA试剂盒进行测定.结果 500 kV变电站工人与对照组的工频电场(t=12.455,P=0.000)、工频磁场(t=18.942,P=0.000)暴露比较差异有统计学意义.经校正后,变电站工人与对照人群外周HSP70蛋白表达水平比较差异无统计学意义(F=0.624,P=0.430).结论 长期接触工频电磁场没有引起变电站工人HSP70表达水平增加.

  9. 高铁作业者血清 HSP70水平分析及护理干预%Investigation and Nursing Interventions on the Expression Level of Serum HSP70 in High-speed Rail Worker

    Institute of Scientific and Technical Information of China (English)

    王宏艳; 徐乐; 叶良平


    目的:探讨高铁作业者血清热应激蛋白70(HSP70)表达水平,为护理干预提供依据。方法收集职业性体检的高铁作业者176名为高温组。从同日体检人群中随机抽取176名为对照组。ELISA 法检测血清 HSP70含量。结果高温组血清HSP70水平(0.157±0.133)ng/ml高于对照组(0.097±0.134) ng/ml ,有显著性差异(t=4.26,P<0.01)。以接毒工龄将高温组分为三组,H S P70含量组间比较,有显著性差异(F=8.26,P<0.01)。结论高铁作业者血清H S P70水平显著升高,可为高温作业制定护理对策提供指导。%Objective To explore the expression level of serum HSP70 of high-speed rail worker .Methods A total of 176 subjects with Heat Stress in high-speed rail worker and the same number of healthy controls in hospital health exami‐nation center were analyzed .The expression levels of serum HSP70 were detected by ELISA .Results The concentrations of Serum HSP70 from Heat Stress group(0 .157 ± 0 .133) ng/ml were higher remarkably than those in the normal control group(0 .097 ± 0 .134) ng/ml ,and there was statistical significance (t=4 .26 ,P<0 .01) .The Heat Stress group was divid‐ed into 3 groups with working -age ,and the difference of the content of HSP70 from 3 groups was statistically significant (F=8 .26 ,P<0 .01) .Conclusions The expression level of serum HSP70 were significantly higher in Heat Stress group than in control group ,and this was to provide guidance for the development of nursing strategies in high-speed rail worker .

  10. Evaluation of Antibody Responses Elicited by Immunization of Mice with a Pneumococcal Antigen Genetically Fused to Murine HSP70 and Murine Interleukin-4

    Institute of Scientific and Technical Information of China (English)

    Dennis O. GOR; Salamatu S. MAMBULA


    The heat shock (stress) protein HSP70 has been shown to be a potent stimulator of cellular immune responses. In order to determine whether HSP70 has the ability to stimulate antibody responses, we constructed and expressed fusion proteins consisting of murine HSP70 or murine interleukin (IL)-4 covalently linked to a pneumococcal cell wall-associated protein antigen designated PpmA. Immunization of mice with the PpmA-HSP70 fusion protein (PpmA-70) failed to elicit an increased PpmA-specific serum antibody response. In contrast, mice immunized with PpmA fused to IL-4 (PpmA-IL4), or PpmA fused to both IL-4 and HSP70 (PpmA-IL4-70) fusion proteins elicited high levels of PpmA-specific antibody responses.These data suggest that HSP70 has a limited capacity to stimulate immune responses to heterologous antigens in vivo.

  11. Impaired brain StAR and HSP70 gene expression in zebrafish exposed to Methyl-Parathion based insecticide. (United States)

    da Rosa, João Gabriel Santos; Koakoski, Gessi; Piato, Angelo L; Bogo, Maurício Reis; Bonan, Carla Denise; Barcellos, Leonardo José Gil


    Fish production ponds and natural water body areas located in close proximity to agricultural fields receive water with variable amounts of agrochemicals, and consequently, compounds that produce adverse effects may reach nontarget organisms. The aim of this study was to investigate whether waterborne methyl-parathion-based insecticide (MPBI) affected gene expression patterns of brain glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR), and heat shock protein 70 (hsp70) in adult zebrafish (Danio rerio) exposed to this chemical for 96 h. Treated fish exposed to MPBI-contaminated water showed an inhibition of brain StAR and hsp70 gene expression. Data demonstrated that MPBI produced a decrease brain StAR and hsp70 gene expression.

  12. [SWI/SNF Protein Complexes Participate in the Initiation and Elongation Stages of Drosophila hsp70 Gene Transcription]. (United States)

    Mazina, M Yu; Nikolenko, Yu V; Krasnov, A N; Vorobyeva, N E


    The participation of the SWI/SNF chromatin remodeling complex in the stimulation of the RNA polymerase II binding to gene promotors was demonstrated in all model eukaryotic organisms. It was shown eight years ago that the SWI/SNF complex influence on transcription is not limited to its role in initiation but also includes participation in elongation and alternative splicing. In the current work, we describe the subunit composition of the SWI/SNF complexes participating in initiation, preparing for the elongation and elongation of hsp70 gene transcription in Drosophila melanogaster. The data reveal the high mobility of the SWI/SNF complex composition during the hsp 70 gene transcription process. We suggest a model describing the process of sequential SWI/SNF complex formation during heat-shock induced transcription of the hsp 70 gene.

  13. Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture: Morphological Changes and Hsp70 Expression

    Institute of Scientific and Technical Information of China (English)


    @@Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

  14. Early Onset of Heat-Shock Response in Mouse Embryos Revealed by Quantification of Stress-Inducible hsp70i RNA

    Directory of Open Access Journals (Sweden)

    Lawrence J. Wangh


    Full Text Available Heat shock response is fully established in mouse embryos at the blastocyst stage, but it is unclear when this response first arises during development. To shed light on this question, we used a single-tube method to quantify mRNA levels of the heat shock protein genes hsp70.1 and hsp70.3 (hsp70i in individual cleavage-stage embryos that had or had not been heat-shocked. While untreated, healthy embryos contained very low copy numbers of hsp70i RNA, heat shock rapidly induced the synthesis of hundreds of hsp70i transcripts per blastomere at both the 4-cell and the 8-cell stages. In addition, we performed hsp70i measurements in embryos that had not been heat-shocked but had been very slow in developing.Quantification of hsp70i RNA and genomic DNA copy numbers in these slow-growing embryos demonstrated the presence of two distinct populations. Some of the embryos contained considerable levels of hsp70i RNA, a finding consistent with the hypothesis of endogenous metabolic stress accompanied by cell cycle arrest and delayed development. Other slow-growing embryos contained no hsp70i RNA and fewer than expected hsp70i gene copies, suggesting the possibility of ongoing apoptosis. In conclusion, this study demonstrates that mouse embryos can activate hsp70i expression in response to sub-lethal levels of stress as early as at the 4-cell stage. Our results also indicate that quantification of hsp70i DNA and RNA copy numbers may provide a diagnostic tool for embryonic health.

  15. HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation. (United States)

    Vasaikar, Suhas V; Ghosh, Sourish; Narain, Priyam; Basu, Anirban; Gomes, James


    Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NFκB determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.

  16. Expression of heat-shock protein 70 (Hsp70) in the respiratory tract and lungs of fire victims. (United States)

    Marschall, S; Rothschild, M A; Bohnert, M


    Immunohistochemical investigation of the respiratory tract and lungs of 63 fire victims revealed a statistically significant enhanced expression of heat-shock protein 70 (Hsp70) in the epiglottis, the trachea, and the main and the peripheral bronchi compared with a control group. In the fire victims, a strong expression of Hsp70 was discernible not only particularly in the vessels but also in seromucous secretory cells, ciliated epithelial cells, smooth muscle cells, and alveolar cells. The results suggest a vital or supravital reaction due to the inhalation of hot fire fumes.

  17. Cloning and procaryotic expression of HSP70 gene of bovine Theileria sergenti%牛瑟氏泰勒虫HSP70基因的克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    金超; 贾立军; 薛书江; 王娜; 钱年超; 张守发


    根据GenBank(D12692.1)上发表的牛瑟氏泰勒虫HSP70基因序列设计、合成1对特异性的引物,采用PCR方法扩增牛瑟氏泰勒虫HSP70基因片段,将扩增产物与pMD18-T simple载体连接,重组质粒经PCR、酶切鉴定后测序;用pGEX-4T-1表达栽体构建重组质粒pGEX-HSP70,经IPTG诱导表达后进行SDS-PAGE、Western-blot分析.结果表明:扩增的HSP70基因与D12692.1序列同源性为99.3%;表达的融合蛋白分子质量约为68 ku,而且可与牛瑟氏泰勒虫阳性血清发生特异性反应.

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

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


    A study was conducted to assess the impact of heat and nutritional stress simultaneously on the adaptive capability as indicated by behavioral and physiological responses, plasma heat shock protein 70 (HSP70) level, and peripheral blood mononuclear cells (PBMC) HSP70 gene expression in goats. Twenty-four adult Osmanabadi bucks (average body weight (BW) 16.0 kg) were used in the present study. The bucks were divided into four groups viz., C ( n = 6; control), HS ( n = 6; heat stress), NS ( n = 6; nutritional stress), and CS ( n = 6; combined stress). The study was conducted for a period of 45 days. C and HS bucks had ad libitum access to their feed while NS and CS bucks were under restricted feed (30 % intake of C bucks) to induce nutritional stress. The HS and CS bucks were exposed to solar radiation for 6 h a day between 10:00 a.m. and 4:00 p.m. to induce heat stress. The data was analyzed using repeated measures analysis of variance. The standing time differed significantly ( P adaptive capabilities of Osmanabadi bucks as compared to that would occur individually. Further, the study indicated that lying time, drinking frequency, RR, RT, plasma HSP70, and PBMC HSP70 gene expression may act as ideal biological markers for assessing the impact of CS on adaptive capabilities in bucks.

  19. 高温工人HSP70基因多态性与高血压易感性%Relationship between single nucleotide polymorphisms of HSP70 genes and susceptibility to hypertension in workers exposed to heat stress

    Institute of Scientific and Technical Information of China (English)

    李瑞芳; 孙建娅; 张萍; 郑金平


    目的 探讨HSP70基因多态性与高温作业工人高血压发病易感性关系.方法 选取某钢铁企业158名炼钢、轧钢工人为高温组,同厂的76名库工为对照组.使用统一的调查表收集个人信息并测定血压值,聚合酶链式反应-限制性片断长度多态性(PCR - RFLP)方法分析HSP70基因多态性,运用PHASE 2.0遗传分析软件计算单倍型.结果 HSP70各基因型在高温组与对照组及高血压与非高血压人群中的分布差异均无统计学意义(P>0.05).进一步将研究对象分为正常血压组、血压高值组和高血压组后发现,高温组血压高值组明显高于高血压组(P<0.05);高温组和对照组2组合并后的总人群中,具有C/C基因型的人群与具有G/G基因型的人群比较,易感血压高值的调整OR(95%CI)为3.52(1.02 ~12.11).高温组血压高值组HSP70 -2基因G/G基因型频率明显高于高血压组及正常血压组,而HSP70 -2基因G/A基因型在血压高值组的分布频率低于高血压组与正常血压组;经Logistic回归分析,高温组具有HSP70-2基因G/A基因型的人群与G/G基因型的人群比较,易感血压高值的调整OR(95%CI)为0.25(0.08~0.78).单倍型对分析,高温组具有GAT/***单倍型的人群与GAT/GAT单倍型对的人群比较,易感高血压的调整OR(95%CI)为1.29(1.02 ~12.44).结论 HSP70 -1基因C/C基因型与人群血压高值有关联.HSP70 -2基因G/G基因型与高温作业工人血压高值有关联,G/A基因型可降低高温作业工人血压高值的风险性,GAT/***单倍型与高温作业工人高血压的易感性有关.%Objective To study the relationship between HSP70 polymorphisms and susceptibility to hypertension in workers exposed to heat stress. Methods Totally 158 steel workers and rollers were selected as the exposure group and 76 workers without heat stress exposure were selected as the control group. The general status and the blood pressure were investigated. HSP70

  20. The Effects of Dimethoate on HSP70 Expression and AChE Activity in Glioma U87 Cells%乐果对U87细胞HSP70表达及AChE活性的影响

    Institute of Scientific and Technical Information of China (English)

    谢芳; 周志俊; 吴强恩; 吴同俊


    [目的]研究乐果对神经胶质瘤细胞U87热应激蛋白70(HSP70)表达的影响规律,比较HSP70与乙酰胆碱酯酶(AChE)对乐果的敏感性.[方法]以不同浓度乐果(100、10、1、0.1、0.01、0 μmol/L)对体外培养的U87细胞分别进行2、4、8、16和24h染毒,再分别用Western-blot法和DTNB法检测各组细胞HSP70表达和AChE活性的变化,比较这两个指标变化的幅度以及引起此变化所需乐果浓度和染毒时间的差异.[结果]乐果染毒2 h即可引起HSP70表达的显著增高(P<0.01),这一变化在染毒4 h时出现于各剂量组,并可持续至染毒16 h.在染毒2 h和4 h,HSP70表达的变化与乐果剂量之间存在相关关系(Pearson相关系数分别为0.916和0.989,P<0.01).AChE活性的显著降低出现在染毒4 h,且仅出现在剂量较高的3个组,变化幅度也较小.[结论]乐果可以在相对较短时间内以较低剂量引起U87细胞HSP70表达的显著变化,并存在一定的剂量-反应关系.HSP70比AChE对乐果更为敏感.

  1. The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels. (United States)

    Young, Jason C


    Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease--the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.

  2. Reactive oxygen species generated by a heat shock protein (Hsp) inducing product contributes to Hsp70 production and Hsp70-mediated protective immunity in Artemia franciscana against pathogenic vibrios. (United States)

    Baruah, Kartik; Norouzitallab, Parisa; Linayati, Linayati; Sorgeloos, Patrick; Bossier, Peter


    The cytoprotective role of heat shock protein (Hsp70) described in a variety of animal disease models, including vibriosis in farmed aquatic animals, suggests that new protective strategies relying upon the use of compounds that selectively turn on Hsp genes could be developed. The product Tex-OE® (hereafter referred to as Hspi), an extract from the skin of the prickly pear fruit, Opuntia ficus indica, was previously shown to trigger Hsp70 synthesis in a non-stressful situation in a variety of animals, including in a gnotobiotically (germ-free) cultured brine shrimp Artemia franciscana model system. This model system offers great potential for carrying out high-throughput, live-animal screens of compounds that have health benefit effects. By using this model system, we aimed to disclose the underlying cause behind the induction of Hsp70 by Hspi in the shrimp host, and to determine whether the product affects the shrimp in inducing resistance towards pathogenic vibrios. We provide unequivocal evidences indicating that during the pretreatment period with Hspi, there is an initial release of reactive oxygen species (hydrogen peroxide and/or superoxide anion), generated by the added product, in the rearing water and associated with the host. The reactive molecules generated are the triggering factors responsible for causing Hsp70 induction within Artemia. We have also shown that Hspi acts prophylactically at an optimum dose regimen to confer protection against pathogenic vibrios. This salutary effect was associated with upregulation of two important immune genes, prophenoloxidase and transglutaminase of the innate immune system. These findings suggest that inducers of stress protein (e.g. Hsp70) are potentially important modulator of immune responses and might be exploited to confer protection to cultured shrimp against Vibrio infection.

  3. hELP3 Subunit of the Elongator Complex Regulates the Transcription of HSP70 Gene in Human Cells

    Institute of Scientific and Technical Information of China (English)

    Qiuju HAN; Xiaozhe HOU; Dongmei SU; Lina PAN; Jizhou DUAN; Liguo CUI; Baiqu HUANG; Jun LU


    The human Elongator complex is remarkably similar to its yeast counterpart in several aspects.In a previous study, we analyzed the functions of the human elongation protein 3 (hELP3) subunit of the human Elongator by using an in vivo yeast complementation system. However, direct evidence for hELP3 functions in regulating gene expression in human cells was not obtained. In this study, we used hELP3 antisense oligonucleotide inhibitors to knock down hELP3 gene expression to investigate its function in human 293T cells. The results showed that specific reduction of hELP3 mRNA and protein caused a significant suppression of HSP70-2 gene expression, and this was accompanied by histone H3 hypoacetylation and decreased RNA polymerase Ⅱ density at the HSP70-2 gene. Moreover, the data also showed that hELP3 exerted the transcriptional regulatory function directly through its presence on the HSP70-2 gene. Data presented in this report provide further insight and direct evidence of the functions of hELP3 in HSP70-2 gene transcriptional elongation in human cells.

  4. Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors. (United States)

    Tukaj, Stefan; Bisewska, Joanna; Roeske, Katarzyna; Tukaj, Zbigniew


    The objective of this study was to examine the influence of different stressors, including cadmium (heavy metal), anthracene (polycyclic aromatic hydrocarbon-PAH) and chloridazon (herbicide), on population growth and biosynthesis of cytoplasmic HSP70 in Lemna minor (duckweed) in short (4 h)- and long (7 days)-term tests. A heat shock response was confirmed in Lemna exposed to high temperature: 35, 37.5, 40, or 42.5°C in short-term (4 h) treatments. The chemicals tested stimulated the biosynthesis of the cytoplasmic HSP70 protein in a concentration-dependent way (0.5-5 μM), higher in fronds exposed to lower doses of stressors. Additionally, production of HSP70 was greater after 4 h of incubation than after 7 days. The results suggest that HSP70 could be applied as a non-specific and sensitive detector of stress induced by different chemicals at concentrations below those that produce the type of response observed in classical cytotoxicity tests, such as growth inhibition.

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

    DEFF Research Database (Denmark)

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


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

  6. Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70

    DEFF Research Database (Denmark)

    Chen, S; Prapapanich, V; Rimerman, R A;


    mature PR complexes. In the present study we observe that a monoclonal antibody specific for p60 can, on the one hand, inhibit formation of mature PR complexes containing heat shock protein 90 (hsp90), p23, and immunophilins and, on the other, enhance recovery of early PR complexes containing hsp70...

  7. Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer. (United States)

    Jönsson, K Ingemar; Schill, Ralph O


    The physiology and biochemistry behind the extreme tolerance to desiccation shown by the so-called anhydrobiotic animals represents an exciting challenge to biology. The current knowledge suggests that both carbohydrates and proteins are often involved in protecting the dry cell from damage, or in the repair of induced damage. Tardigrades belong to the most desiccation-tolerant multicellular organisms, but very little research has been reported on the biochemistry behind desiccation tolerance in this group. We quantified the induction of the heat-shock protein Hsp70, a very wide-spread stress protein, in response to desiccation, ionising radiation, and heating, in the anhydrobiotic tardigrade Richtersius coronifer using an immuno-westernblot method. Elevated levels of Hsp70 were recorded after treatment of both heat and ionising radiation, and also in rehydrated tardigrades after a period of desiccation. In contrast, tardigrades in the desiccated (dry) state had reduced Hsp70 levels compared to the non-treated control group. Our results suggest that Hsp70 may be involved in the physiological and biochemical system underlying desiccation (and radiation) tolerance in tardigrades, and that its role may be connected to repair processes after desiccation rather than to biochemical stabilization in the dry state.

  8. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70. (United States)

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le


    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.

  9. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation. (United States)

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego


    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  10. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1. (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai


    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  11. Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis. (United States)

    Carrillo, Eugenia; Crusat, Martín; Nieto, Javier; Chicharro, Carmen; Thomas, Maria del Carmen; Martínez, Enrique; Valladares, Basilio; Cañavate, Carmen; Requena, Jose María; López, Manuel Carlos; Alvar, Jorge; Moreno, Javier


    Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis.

  12. Microbiota Composition, HSP70 and Caspase-3 Expression as Marker for Colorectal Cancer Patients in Aceh, Indonesia

    Directory of Open Access Journals (Sweden)

    Fauzi Yusuf


    Full Text Available Aim: to investigate the relationship between microbiota composition with HSP70 and Caspase-3 expressions in colon tissue as an initial study to develop the candidate for early detection of colorectal cancer for Indonesian patients. Methods: this is a cross-sectional study on 32 patients undergoing colonoscopy; 16 patients of colorectal cancer (CRC while the other 16 patients are not (colitis and internal hemorrhoid. The composition of microbiota in stool samples was examined using 16S rRNA Denaturing Gradient Gel Electrophoresis (DDGE while expression of HSP70 was examined by immunohistochemistry and Caspase-3 by using Haematoxylin-Eosin(HE staining to determine the morphological changes in colon tissue. Results: analysis of PCR-DDGE shows a different composition of microbiota between patients with CRC and non-CRC. All CRC patients showed disappearance of dominant band from Bifidobacterium groups. Histological observation based on Inter Class Correlation (ICC test from all slide showed a high scores (5.2-9.2 in CRC patients and low scores (1.7-2.4 in non-CRC patients. HSP70 expression was increased significantly in CRC patients with the highest percentage of 84%, while expression of caspase-3 decreased with the highest percentage of 21%. Statistical analysis showed that the incidence of colorectal cancer was associated with the expression of HSP 70 (p<0.001, and Caspase 3 (p<0.001. Conclusion: bifidobacterium is an important indicator for colorectal cancer patients that show disappearance of dominant band, while expression of HSP70 increased and the Caspase-3 expression decreased significantly.

  13. Expression of Hsp27 and Hsp70 in lymphocytes and plasma in healthy workers and coal miners with lung cancer. (United States)

    Wang, Haijiao; Xing, Jingcai; Wang, Feng; Han, Wenhui; Ren, Houmao; Wu, Tangchun; Chen, Weihong


    In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. We investigated the association between levels of Hsp27 and Hsp70 expression in lymphocytes and plasma and levels of coal-mine dust exposure in workplace or risk of lung cancer in 42 cancer-free non-coal miners, 99 cancer-free coal miners and 51 coal miners with lung cancer in Taiyuan city in China. The results showed that plasma Hsp27 levels were increased in coal miners compared to non-coal miners (P<0.01). Except high cumulative coal-mine dust exposure (OR=13.62, 95%CI=6.05-30.69) and amount of smoking higher than 24 pack-year (OR=2.72, 95% CI=1.37-5.42), the elevated levels of plasma Hsp70 (OR=13.00, 95% CI=5.14-32.91) and plasma Hsp27 (OR=2.97, 95% CI=1.40-6.32) and decreased expression of Hsp70 in lymphocytes (OR=2.36, 95% CI=1.05-5.31) were associated with increased risk of lung cancer. These findings suggest that plasma Hsp27 may be a potential marker for coal-mine dust exposure. And the expression of Hsp27 and Hsp70 levels in plasma and lymphocytes may be used as biomarkers for lung cancer induced by occupational coal-mine dust exposure.

  14. Effects of cold stress on HSP70 mRNA expression in intestines of chickens%冷应激对雏鸡肠组织中HSP70mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    于宪一; 李术; 陈蕾; 张雯; 毕明玉; 唐洪鹏; 张子威; 徐铭; 徐世文


    探讨冷应激对雏鸡肠道中热休克蛋白70(HSP70)mRNA表达量的影响.120只1日龄的伊莎公鸡,饲养至15日龄,随机平均分为12组,在(12±1)℃进行急性和慢性冷应激处理.急性冷应激时间为0,1,3,6,12和24 h;慢性冷应激时间为5、10和20 d,并各设对照组.各时间点随机剖杀5只,取十二指肠、空肠、回肠和盲肠用实时定量PCR法检测HSP70mRNA的表达.结果表明,急性应激时,各组织中HSP70mRNA的表达整体降低,但在初期显著升高(P<0.05).慢性冷应激时,各组织中HSP70mRNA整体显著升高(P<0.05).结果显示,冷应激可以改变雏鸡肠组织中HSP70mRNA的表达,表现为急性冷应激初期和慢性冷应激时,肠组织中HSP70mRNA表达量增加,而急性冷应激后期下降.%In this study, the effects of cold stress on the expression levels of HSP70 in duodenum, jejunum, ileum and cecum were studied. 120 1-day-old male chickens were kept under the temperature of (30±2) ℃ and were given commercial diet and water. When the chickens were 15 days old, they were randomly divided into 12 groups on average and were kept under the temperature of (12±1) ℃ for acute and chronic cold stress. The duration of acute cold stress were 0, 1, 3, 6, 12 and 24 h, and the duration of chronic cold stress were 5, 10 and 20 d. 0 h was the control group for the acute cold stress, and there were three control groups for chronic cold stress. After the cold stress treatment, the tissues were collected for real-time PCR assessment. The results showed that at the beginning of the acute cold stress and chronic cold strss, HSP70 expression levels were increased significantly(P<0.01), but at the end of acute cold stress, the expression levels were decreased significantly(P<0.01). The study suggested that HSP70 expressioin levels could be influenced by the cold stress and the expression levels be different by different tissues or different durations of the cold exposure.

  15. Differential role for ERK2 in anoxia-induced activation of transcription and translation of Hsp70 in NIH 3T3 cells

    DEFF Research Database (Denmark)

    Ossum, Carlo; Lauritsen, Anders N.; Karottki, Dorina Gabriela;


    and transcription was involved. Inhibition of the MAP kinase p38, which was transiently activated during chemical anoxia, had no effect on the increase in Hsp70 expression whereas an inhibitor of reactive oxygen species and inhibition of the phosphatase PP1 and PP2a inhibited the increase in Hsp70 expression...

  16. Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p. (United States)

    Sharma, Deepak; Masison, Daniel C


    Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.

  17. 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.;


    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...

  18. BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP. (United States)

    Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris


    Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc.

  19. Induction of heat shock protein 70 (Hsp70) by proteasome inhibitor MG 132 protects articular chondrocytes from cellular death in vitro and in vivo. (United States)

    Grossin, Laurent; Etienne, Stéphanie; Gaborit, Nadège; Pinzano, Astrid; Cournil-Henrionnet, Christel; Gerard, Catherine; Payan, Elisabeth; Netter, Patrick; Terlain, Bernard; Gillet, Pierre


    The aim of this work was to determine whether Hsp70 overexpression via proteasome inhibitor MG132 was able to protect chondrocytes towards mono-iodoacetate (MIA) cytotoxicity both in vitro and in vivo. In vitro, overexpression of Hsp70 via MG132 was significantly able to protect chondrocytes from MIA toxicity (MTT/LDH analyses). Hsp70 essentially mediated this chondroprotective effect as demonstrated by antisense strategy. In vivo, chondrocytic overexpression of Hsp70, after a preventive intra-articular injection of MG132 in rat knee, was sufficient to decrease the severity of OA-induced MIA lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of Hsp70, through proteasome inhibition, could be an interesting tool in protecting chondrocytes from cellular injuries, either necrotic or apoptotic in nature, and thus might be a novel chondroprotective modality in rat experimental OA.

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

    Energy Technology Data Exchange (ETDEWEB)

    Haap, Timo, E-mail:; Schwarz, Simon; Köhler, Heinz-R.


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

  1. 基于Hsp70基因的马梨形虫分类学定位分析%Taxonomic Status Assays of Theileria equi Based on Hsp70 Gene

    Institute of Scientific and Technical Information of China (English)

    罗金; 刘光远; 田占成; 谢俊仁; 张萍; 沈辉; 党根生


    [Objective] The objective of the study is to determine the taxonomic status of Theileria equi (T. equi). [Method] According to the Hsp70 gene sequence (AB248743.1) of Babesia equi (B.equi) in GenBank, the primers TeHsp70F and TeHsp70R were designed. And a fragment with the length of 1 920 bp was obtained by PCR. The gene sequence was compared with other 23 amino acids sequences. [Result] The fragment encods 639 amino acids, 208 hydrophobic amino acids and 167 polar amino acid. Identity analysis showed that T. equi has a closest relationship with B.equi (BAF0262S.1), and has a little relationship with T. parva (XP764717.1) and T. annulata (AAA30130.1), while T. equi has a distant relationship with B. caballi (BAF02619.1), which infects equine. [ Conclusion ] It was a correct taxonomic status for B. equi to Theileria species. And the taxonomic status was error before, for T. equi to Babesia species. In this paper, a proof was provided for taxonomic status of Theileria equi.%[目的]为了进一步确定马泰勒虫的分类学定位.[方法]根据GenBank上登录的马巴贝斯虫Hsp70基因序列(AB248743.1),设计引物TeHsp70F,TeHsp70R,以马泰勒虫基因组DNA为模板进行扩增,获得全长为1920 bp的核酸片段.将该基因序列与GenBank中23种已知虫种的相应序列进行分析比较.[结果]该片段编码639个氨基酸,其疏水性氨基酸达到208个,极性氨基酸167个.同一性分析显示,该基因片段与报道的马巴贝斯虫Hsp70基因(B.equi BAF02625.1)亲缘关系最近,其次是小泰勒虫(T.parva XP764717.1)和环形泰勒虫(T.annulataAAA30130.1),而与感染马属动物的另一个虫种驽巴贝斯虫(B.caballi BAF02619.1)亲缘关系较远.[结论]马泰勒虫Hsp70基因的克隆及系统发育分析显示,之前被定名为马巴贝斯虫(B.equi)的虫种隶属于泰勒虫虫种.本试验为马巴贝斯虫正确更名为马泰勒虫(T.equi)的分类地位提供了又一佐证.

  2. Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells. (United States)

    Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D


    Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

  3. Magnetic thermoablation stimuli alter BCL2 and FGF-R1 but not HSP70 expression profiles in BT474 breast tumors

    Directory of Open Access Journals (Sweden)

    Stapf M


    Full Text Available Marcus Stapf, Nadine Pömpner, Melanie Kettering, Ingrid Hilger Institute of Diagnostic and Interventional Radiology, Jena University Hospital, Jena, Germany Abstract: Magnetically induced heating of magnetic nanoparticles (MNP in an alternating magnetic field (AMF is a promising minimal invasive tool for localized tumor treatment that eradicates tumor cells by applying thermal stress. While temperatures between 42°C and 45°C induce apoptosis and sensitize the cells for chemo- and radiation therapies when applied for at least 30 minutes, temperatures above 50°C, so-called thermoablative temperatures, rapidly induce irreversible cell damage resulting in necrosis. Since only little is known concerning the protein expression of anti-apoptotic B-cell lymphoma 2 (BCL2, fibroblast growth factor receptor 1 (FGF-R1, and heat shock protein (HSP70 after short-time magnetic thermoablative tumor treatment, these relevant tumor proteins were investigated by immunohistochemistry (IHC in a human BT474 breast cancer mouse xenograft model. In the investigated sample groups, the application of thermoablative temperatures (<2 minutes led to a downregulation of BCL2 and FGF-R1 on the protein level while the level of HSP70 remained unchanged. Coincidently, the tumor tissue was damaged by heat, resulting in large apoptotic and necrotic areas in regions with high MNP concentration. Taken together, thermoablative heating induced via magnetic methods can reduce the expression of tumor-related proteins and locally inactivate tumor tissue, leading to a prospectively reduced tumorigenicity of cancerous tissues. The presented data allow a deeper insight into the molecular mechanisms in relation to magnetic thermoablative tumor treatments with the aim of further improvements. Keywords: magnetic nanoparticles (MNP, thermoablation, in vivo, mouse model, breast cancer tumor

  4. 汉坦病毒诱导人脐静脉内皮细胞HSP70的表达及意义

    Institute of Scientific and Technical Information of China (English)

    余璐; 马恒; 刘彦仿; 杨守京


    目的:研究人脐静脉内皮细胞(HUVEC)感染汉坦病毒(Hantavirus,HTV)后应激反应的规律及意义.方法:采用免疫细胞化学染色法和核酸分子原位杂交,检测热休克蛋白70(HSP70)的表达;用RT-PCR观察HSP70 mRNA水平变化的规律.结果:HTV感染HUVEC后,免疫细胞化学染色法可检出HSP70呈高表达,原位杂交发现细胞质内有HSP70 mRNA的阳性信号.感染后不同时间点RT-PCR的结果表明,与对照组相比较,HSP70 mRNA的水平在感染后迅速升高并持续高表达(P<0.05).结论:HTV可诱导HUVEC高表达HSP70HSP70可能具有抑制病毒复制和保护内皮细胞的作用.

  5. Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    DAI; Wuxing; HUANG; Hailang; YUAN; Ye; HU; Jiajie; HUANGFU; Yongmu


    The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index(SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ(IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0. 01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0. 01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0. 05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0. 05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

  6. Effects of broad band electromagnetic fields on HSP70 expression and ischemia-reperfusion in rat hearts. (United States)

    Ronchi, Raffaella; Marano, Lidia; Braidotti, Paola; Bianciardi, Paola; Calamia, Mario; Fiorentini, Cesare; Samaja, Michele


    Although exposure to broad band (0.2-20 MHz) electromagnetic fields (EMF) is part of the treatment of several diseases, little is known as to their effects on myocardial protein expression and resistance to ischemia-reperfusion (I/R). We exposed Sprague-Dawley rats to either high (H, 10 min/day at 200 V/m, 36.1 microT) or low (L, 2 min/day at 30 V/m, 11.4 microT) intensity broad band EMF for 15 days. At the end of the treatment, myocardial HSP70 was 32 +/- 8% (mean +/- SEM) higher in L (P = 0.01) than in control (C), whereas in H it remained the same as in C. Electron microscopy revealed sporadic ruptures of mitochondrial cristae in H hearts, with no differences in other parameters. Malondialdehyde was increased in treated hearts (P < 0.05), but especially in H (P = 0.008). To assess the protective role of HSP70 during I/R, hearts were Langendorff-perfused with Krebs-Henseleit. After I/R, C hearts displayed depressed rate. pressure (-13 +/- 7%) and increased end-diastolic (+9.2 +/- 2.8 mmHg) and perfusion pressures (+30 +/- 10 mmHg). In H and L, rate. pressure recovery was similar to C (-2 +/- 21% and -12 +/- 16%, respectively, P = NS). In contrast, both end-diastolic and perfusion pressures were higher in L than in H (30.8 +/- 5.4 vs 18.2 +/- 3.5, P = 0.01, and 54 +/- 8 vs 21 +/- 8 mmHg, P = 0.01, respectively) indicating diastolic derangement in L. In conclusion, the effects of broad band EMF on HSP70 appear to be biphasic, and HSP70 overexpression might not be directly related to improved protection against I/R.

  7. Bleaching and stress in coral reef ecosystems: hsp70 expression by the giant barrel sponge Xestospongia muta. (United States)

    López-Legentil, Susanna; Song, Bongkeun; McMurray, Steven E; Pawlik, Joseph R


    Sponges are a prominent component of coral reef ecosystems. Like reef-building corals, some sponges have been reported to bleach and die. The giant barrel sponge Xestospongia muta is one of the largest and most important components of Caribbean coral reef communities. Tissues of X. muta contain cyanobacterial symbionts of the Synechococcus group. Two types of bleaching have been described: cyclic bleaching, from which sponges recover, and fatal bleaching, which usually results in sponge death. We quantified hsp70 gene expression as an indicator of stress in X. muta undergoing cyclic and fatal bleaching and in response to thermal and salinity variability in both field and laboratory settings. Chlorophyll a content of sponge tissue was estimated to determine whether hsp70 expression was related to cyanobacterial abundance. We found that fatally bleached sponge tissue presented significantly higher hsp70 gene expression, but cyclically bleached tissue did not, yet both cyclic and fatally bleached tissues had lower chlorophyll a concentrations than nonbleached tissue. These results corroborate field observations suggesting that cyclic bleaching is a temporary, nonstressful state, while fatal bleaching causes significant levels of stress, leading to mortality. Our results support the hypothesis that Synechococcus symbionts are commensals that provide no clear advantage to their sponge host. In laboratory experiments, sponge pieces incubated at 30 degrees C exhibited significantly higher hsp70 expression than control pieces after 1.5 h, with sponge mortality after less than 15 h. In contrast, sponges at different salinities were not significantly stressed after the same period of time. Stress associated with increasing seawater temperatures may result in declining sponge populations in coral reef ecosystems.

  8. Effects of rapid temperature changes on HK, PK and HSP70 of Litopenaeus vannamei in different seasons (United States)

    Guo, Biao; Wang, Fang; Dong, Shuanglin; Hou, Chunqiang


    Activities of hexokinase (HK), pyruvate kinase (PK) and levels of HSP70 were measured to evaluate the response of Litopenaeus vannamei to rapid temperature changes under controlled laboratory conditions. Shrimps were subjected to a quick temperature change from 27°C to 17°C for the summer case (Cold temperature treatment), or from 17°C to 27°C for the winter case (Warm temperature treatment). After 0.5, 1, 3, 6, 12, 24, 48, and 72 h of exposure time, shrimps were sampled and prepared for further analysis. The results showed that the effect of acute temperature changes on activities of HK was significant. Patterns of variations of the two glycolytic enzymes suggested that enzymes in the glycolysis cycle could adjust their activities to meet the acute temperature change. The HSP70 level increased in both cold and warm temperature treatments, suggesting that the rapid temperature changes activated the process of body’s self-protection. But the difference in expression peak of HSP70 might be related to the different body size and the higher thermal sensitivity to temperature increase than to temperature decrease of L. vannamei.

  9. 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


    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.

  10. Physiological plasticity related to zonation affects hsp70 expression in the reef-building coral Pocillopora verrucosa (United States)

    Poli, Davide; Fabbri, Elena; Goffredo, Stefano; Airi, Valentina


    This study investigates for the first time the transcriptional regulation of a stress-inducible 70-kDa heat shock protein (hsp70) in the scleractinian coral Pocillopora verrucosa sampled at three locations and two depths (3 m and 12 m) in Bangka Island waters (North Sulawesi, Indonesia). Percentage of coral cover indicated reduced habitat suitability with depth and at the Tanjung Husi (TA) site, which also displayed relatively higher seawater temperatures. Expression of the P. verrucosa hsp70 transcript evaluated under field conditions followed a depth-related profile, with relatively higher expression levels in 3-m collected nubbins compared to the 12-m ones. Expression levels of metabolism-related transcripts ATP synthase and NADH dehydrogenase indicated metabolic activation of nubbins to cope with habitat conditions of the TA site at 3 m. After a 14-day acclimatization to common and fixed temperature conditions in the laboratory, corals were subjected for 7 days to an altered thermal regime, where temperature was elevated at 31°C during the light phase and returned to 28°C during the dark phase. Nubbins collected at 12 m were relatively more sensitive to thermal stress, as they significantly over-expressed the selected transcripts. Corals collected at 3 m appeared more resilient, as they showed unaffected mRNA expressions. The results indicated that local habitat conditions may influence transcription of stress-related genes in P. verrucosa. Corals exhibiting higher basal hsp70 levels may display enhanced tolerance towards environmental stressors. PMID:28199351

  11. Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.

    Directory of Open Access Journals (Sweden)

    Francesco A Aprile

    Full Text Available Oligomerization in the heat shock protein (Hsp 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.

  12. Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages. (United States)

    Schill, Ralph O; Steinbrück, Günther H B; Köhler, Heinz-R


    The eutardigrade Milnesium tardigradum can undergo cryptobiosis, i.e. entry into a reversible ametabolic stage induced by dehydration, cooling and, probably, osmotic and anoxic stress. For the first time in tardigrades, we described partial sequences of three heat-shock protein (hsp70 family) genes and examined gene expression on the way from an active to a cryptobiotic and back to an active stage again. Results showed different patterns of gene expression in the hsp70 isoforms. All three isoforms seem to be true heat-shock proteins since transcription could be clearly enhanced by temperature elevation. Isoform 1 and, at a lower level, isoform 3 do not seem to have a specific function for cryptobiosis. By contrast, transcription of isoform 2 is significantly induced in the transitional stage between the active and the cryptobiotic stage, resulting in a comparatively high mRNA copy number also during cryptobiosis. This pattern of induction implies that isoform 2 is the most relevant hsp70 gene for M. tardigradum individuals entering the cryptobiotic stage.

  13. Effects of heated hydrotherapy on muscle HSP70 and glucose metabolism in old and young vervet monkeys. (United States)

    Kavanagh, Kylie; Davis, Ashely T; Jenkins, Kurt A; Flynn, D Mickey


    Increasing heat shock protein 70 (HSP70) in aged and/or insulin-resistant animal models confers benefits to healthspan and lifespan. Heat application to increase core temperature induces HSPs in metabolically important tissues, and preliminary human and animal data suggest that heated hydrotherapy is an effective method to achieve increased HSPs. However, safety concerns exist, particularly in geriatric medicine where organ and cardiovascular disease commonly will preexist. We evaluated young vervet monkeys compared to old, insulin-resistant vervet monkeys (Chlorocebus aethiops sabaeus) in their core temperatures, glucose tolerance, muscle HSP70 level, and selected safety biomarkers after 10 sessions of hot water immersions administered twice weekly. Hot water immersion robustly induced the heat shock response in muscles. We observed that heat-treated old and young monkeys have significantly higher muscle HSP70 than control monkeys and treatment was without significant adverse effects on organ or cardiovascular health. Heat therapy improved pancreatic responses to glucose challenge and tended to normalize glucose excursions. A trend for worsened blood pressure and glucose values in the control monkeys and improved values in heat-treated monkeys were seen to support further investigation into the safety and efficacy of this intervention for metabolic syndrome or diabetes in young or old persons unable to exercise.

  14. Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain (United States)

    Aprile, Francesco A.; Dhulesia, Anne; Stengel, Florian; Roodveldt, Cintia; Benesch, Justin L. P.; Tortora, Paolo; Robinson, Carol V.; Salvatella, Xavier; Dobson, Christopher M.; Cremades, Nunilo


    Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. PMID:23840795

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

    Directory of Open Access Journals (Sweden)

    Deepak Sharma

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

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

    Directory of Open Access Journals (Sweden)

    Li Y


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

  17. 二硫化碳接触工人热应激蛋白70及其抗体水平的变化%Changes of heat stress protein 70 (HSP70) level and the titer of serum antibody to HSP70 in workers exposed to carbon disulfide (SO2)

    Institute of Scientific and Technical Information of China (English)

    王红; 毕勇毅; 文冠华; 陈胜; 肖成峰; 邬堂春


    目的探讨二硫化碳诱导人体产生热应激蛋白70(HSP 70)及其抗体水平情况.方法用免疫印迹法(Western blot)和酶联免疫吸附(ELISA)法对职业性接触二硫化碳工人HSP 70HSP 70抗体水平进行检测.结果接触组工人中血浆HSP 70抗体阳性率明显高于对照组(P<0.05),而HSP 70蛋白含量在两组间比较差异没有显著性(P>0.05);但据HSP 70抗体是否为阳性将工人分为阳性组和阴性组后,阳性组HSP 70蛋白含量低于阴性组,差异有显著性(P<0.05).结论 HSP 70及其抗体滴度可反映二硫化碳的接触状况,对接触二硫化碳作业工人的健康监护有一定意义.

  18. 内源性大麻素2-AG促进HSP70基因的热应激表达%Endocannabinoid 2-AG enhances HSP70 gene expression in heat stress response in vitro

    Institute of Scientific and Technical Information of China (English)

    吴娟; 屈明玥; 张伟; 王登高; 王颖莹


    Objectives To investigate the regulation role of endocannabinoid in heat stress response and its possible receptor signaling pathway. Methods Rat glioma C6 cells were pretreated with different concentra-tions (10-7, 10-6 and 10-5mol/L) of endocannabinoid 2-Arachidonylglycerol (2-AG) for 1 h, then heat stress (42 ℃ water bath) was performed for 1 h. Reverse transcription-PCR and Western blotting were used to detect the expression levels of HSP70 mRNA and protein. The blockade effects of selective CB1 antagonist AM251 (10-4mol/L) or CB2 antagonist AM630 (10-4mol/L) were also observed by adding them to the culture medi-um when 2-AG was pretreated. Results The expression levels of HSP70 mRNA and protein in heat stressed C6 cells were significantly increased after 2-AG treatment. This enhancement was mainly through CB1 receptor signaling pathway. Conclusion Certain level of 2-AG significantly promotes the expression of HSP70 gene post heat stress, suggesting that 2-AG is involved in the regulation of heat stress response. The promotion effect of 2-AG in C6 cells is mainly mediated through CB1 receptor signaling pathway, but not CB2.%目的 探讨内源性大麻素对热应激反应是否存在调控作用,及其可能的受体调控机制.方法 以不同浓度(10-7、10-6、10-5mol/L)内源性大麻素2-AG(2-Arachidonylglycerol)预处理大鼠胶质瘤C6细胞1 h,热刺激(42℃水浴)1 h,通过RT-PCR及Western blot检测热休克蛋白70(heat shock protein 70,HSP70)的mRNA及蛋白表达水平;同时观察特异性受体拮抗剂AM251或AM630的拮抗效应,探讨信号通路机制.结果 内源性大麻素2-AG可显著促进热刺激处理的C6细胞HSP70基因及蛋白的表达,该调控效应由CB1介导.结论 一定浓度的内源性大麻素2-AG对热刺激处理的C6细胞内HSP70基因的表达有明显促进效应,表明2-AG对细胞热应激反应可能具有一定的调控能力;同时,在C6细胞内,该调控效应主要由CB1介导.

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

    Directory of Open Access Journals (Sweden)

    Nguyen Hong Loc

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

  20. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

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    Lei DONG


    Full Text Available Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA and heat shock protein 70 (HSP70 in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each: PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS. After immunization with recombinant proteins, the PSCA-specific cellular immune responses were monitored with ELISPOT, intracellular cytokine staining assay, and flow cytometry, and ELISA assay was used to detect humoral immune responses. The tumor growth and survival of vaccined mice were observed. Results ELISPOT revealed that the mice in PSCA-HSP group generated much more IFN-γ spot-forming cells than those in other groups (P<0.05, and they could generate strong anti-PSCA antibody response. Results of flow cytometry showed that the number of CD8+/IFN-γ+ T cells was significantly higher in PSCAHSP group than that in other groups (P<0.05. ELISA results revealed that all the mice in PSCA, PSCA+HSP and PSCA-HSP group were induced to generate the PSCA-specific humoral immune response, and no statistical difference was found on the antibody levels among the three groups. Animal experiment showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. Conclusion HSP70 is a chaperone with significant effect for protein vaccines, and the recombinant fusion protein PSCA-HSP70 could be of potential value for prostate cancer treatment. DOI: 10.11855/j.issn.0577-7402.2014.09.08

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

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


    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.

  2. Thermal stress in Larvae of the sand dollar (Dendraster excentricus) induces changes in hsp70 gen expression (United States)

    Olivares-Bañuelos, T.; Garcia-Echauri, L.; Figueroa-Flores, S.; Carpizo-Ituarte, E.


    The relationship between temperature and individual performance is reasonably well understood, and much climate-related research has focused on potential shifts in distribution and abundance driven directly by temperature(1). It is known, that global climate change has profound implications for marine ecosystems and the economic and social systems that depend upon them. In virtually all organisms, including equinoderms, cells respond to a variety of stresses, as temperature changes, by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs) (2). Differential Hsp expression among species may allow functionally important genetic variation to cope with thermal stress and other environmental stressors, allowing populations to respond effectively to environmental change. The aim of the present work was to elucidate the effect of different temperatures on hsp70 expression in larvae of the sand dollar Dendraster excentricus. We are interested to understand what is how organisms that live in estuaries respond to global warming. We did thermo tolerance assays whit 3 different larval stages of the sand dollar, including 8 arms plutei, competent and metamorphic larvae. We tested 4 experimental temperatures (4, 20, 26, and 29°C) and 3 different times (1, 3 and 6 hours). After each treatment, total RNA was extracted and used to quantify hsp70 expression levels using real time PCR analysis. We detected, after 6 hours of thermal stress, the highest over-expression levels of hsp70 in both metamorphic and competent larvae. In 8 arms pluetus larvae, no significant differences in hsp70 expression were observed after the exposure to the experimental temperatures. Taking together our results, indicate that larvae of Dendraster excentricus are responding according to their development stage to cope with thermal-stress. The capability of sand dollar larvae to acclimate and respond effectively to the climatic change would be determinated by the sea

  3. Heat Stress Induces Extended Plateau of Hsp70 Accumulation--A Possible Cytoprotection Mechanism in Hepatic Cells. (United States)

    Miova, Biljana; Dinevska-Kjovkarovska, Suzana; Esplugues, Juan V; Apostolova, Nadezda


    The relevance of heat preconditioning resides in its ability to protect cells from different kinds of injury by induction of heat shock proteins, a process in which the intensity of heat stress (HS) and duration of subsequent recovery are vital. This study evaluates the effects of moderate HS (45 min/43°C) and the time-dependent changes during recovery period of HSP70, Bcl-2 and p53 gene and protein expression in HepG2 cells. We also evaluated the effects of 0.4 mM aspirin (ASA) as a potential pharmacological co-inducer of HSP, both alone and in a combination with HS (ASA + HS). HS alone and ASA + HS caused a major up-regulation of HSP70 mRNA in the first 2 h, while HSP70 protein increased gradually and was especially abundant from 2 h to 24 h. Regarding Bcl-2, all treatments rendered similar results: gene expression was down-regulated in the first 2 h, after which there was protein elevation (12-48 h after HS). mRNA expression of p53 in HS- and (ASA + HS)-cells was down-regulated in the first 12 h. The immediate decrease of p53 protein after HS was followed by a biphasic increase. In conclusion, 0.4 mM ASA + HS does not act as a co-inducer of HSP70 in HepG2 cells, but promotes Bcl-2 protein expression during prolonged treatment. Our suggestion is that hepatic cells are most vulnerable in the first 2-6 h, but may have a high capacity for combating stress 12-24 h after HS. Finally, short-term exposure HS might be a "physiological conditioner" for liver cells to accumulate HSP and Bcl-2 proteins and thus obtain cytoprotection against an additional stress.

  4. Expression of Hsp27 and Hsp70 in Lymphocytes and Plasma in Healthy Workers and Coal Miners with Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    王海椒; 邢景才; 王峰; 韩文慧; 任侯卯; 邬堂春; 陈卫红


    In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. ...

  5. Time- and Dose-Dependent Induction of HSP70 in Lemna minor Exposed to Different Environmental Stressors



    The objective of this study was to examine the influence of different stressors, including cadmium (heavy metal), anthracene (polycyclic aromatic hydrocarbon—PAH) and chloridazon (herbicide), on population growth and biosynthesis of cytoplasmic HSP70 in Lemna minor (duckweed) in short (4 h)- and long (7 days)-term tests. A heat shock response was confirmed in Lemna exposed to high temperature: 35, 37.5, 40, or 42.5°C in short-term (4 h) treatments. The chemicals tested stimulated the biosynth...

  6. Adverse effect of tannery waste leachates in transgenic Drosophila melanogaster: role of ROS in modulation of Hsp70, oxidative stress and apoptosis. (United States)

    Siddique, Hifzur R; Gupta, Subash C; Mitra, Kalyan; Bajpai, Virendra K; Mathur, Neeraj; Murthy, Ramesh C; Saxena, Daya K; Chowdhuri, Debapratim K


    Leachate is a complex chemical mixture of chemicals produced as a result of leaching of solid wastes. The potential toxicity of leachates is a major environmental health concern. The present study evaluated the role of ROS in tannery leachates induced Hsp70 expression, antioxidant enzymes and apoptosis in Drosophila. Different concentrations (0.05-2.0%) of leachates prepared from tannery waste at different pH (7.00, 4.93 and 2.88) were mixed with Drosophila food and fed to the larvae for 2-48 h to examine the different stress and apoptotic markers. A concentration- and time-dependent significant increase in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content were observed in the exposed larvae. Activities of antioxidant enzymes were delayed compared with Hsp70 expression and MDA level in the exposed organisms. Apoptotic cell death was observed in the exposed larvae at higher concentrations concurrent with a significant regression in Hsp70 along with a higher level of ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression at these concentrations indicated the important role of ROS in the induction of cellular damage in the exposed organisms. There was a significant generation of ROS in the larvae exposed to 0.5% of leachates which did not interfere with the protection of their cells by Hsp70 and antioxidant enzymes. However, generation of significantly higher levels of ROS in the larvae exposed to 1.0% and 2.0% leachates may decrease Hsp70 expression thus leading to mitochondria-mediated caspase-dependent apoptotic cell death.

  7. Mild heat stress at a young age in Drosophila melanogaster leads to increased Hsp70 synthesis after stress exposure later in life

    Indian Academy of Sciences (India)

    Torsten Nygaard Kristensen; Jesper Givskov Sørensen; Volker Loeschcke


    In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines of Drosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34°C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high temperature treatment known to induce Hsp70 in the investigated species (1 h at 37°ºC). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both ‘mild stress’ and ‘no stress’ flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.

  8. In vitro Study on Role of Hsp70 Expression in DNA Damage of Human Embryonic Lung Cells Exposed to Benzo[a]pyrene

    Institute of Scientific and Technical Information of China (English)



    Objective Benzo[a]pyrene (B[a]P), a ubiquitous environmental pollutant, is a potent procarcinogen and mutagen that can elicit tumors, leading to malignancy. Heat shock proteins (Hsp) have been shown to protect cells against damages caused by various stresses including exposure to numerous chemicals. Whether Hsps, or more specifically Hsp70, are involved in repair of B[a]P-induced DNA damage is currently unknown. Methods We assessed the potential role of the inducible form of Hsp70 in B[a]P-induced DNA damage of human embryonic lung (HEL) cells using immunoblot and the comet assay (i.e., the single cell gel electrophoresis assay). Results Exposure to B[a]P induced a dose-dependent decrease in the level of Hsp70, but a dose-dependent increase in DNA damage both in untreated (control) HEL cells and in cells preconditioned by a heat treatment. Heat preconditioning prior to B[a]P exposure potentiated the effect of B[a]P at a low dose (10 (mol/L), but appeared to be protective at higher doses. There was a negative correlation between Hsp70 level and DNA damage in the non-preheated as well as in the preconditioned cells. Conclusion These data suggest that exposure of HEL cells to B[a]P may induce a dose-dependent reduction in the levels of the inducible Hsp70. The detailed mechanisms for the reduction of Hsp70 levels by B[a]P and the role of Hsp70 in DNA damage under different concentrations of B[a]P remains to be determined.

  9. A cytosolic relay of heat shock proteins HSP70-1A and HSP90β monitors the folding trajectory of the serotonin transporter. (United States)

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H; Freissmuth, Michael; Sucic, Sonja


    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.

  10. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.

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    Ricardo Andrade Zampieri


    Full Text Available Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.Exploring the High Resolution Melting (HRM dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR targeting heat-shock protein 70 coding gene (hsp70 revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania infantum chagasi, L. (L. amazonensis, L. (L. mexicana, L. (Viannia lainsoni, L. (V. braziliensis, L. (V. guyanensis, L. (V. naiffi and L. (V. shawi, and three species found in Eurasia and Africa, including L. (L. tropica, L. (L. donovani and L. (L. major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.

  11. Protective Effects of Focal Ischemic Preconditioning and HSP70 Expression on Middle Cerebral Artery Occlusion in Rats

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jianhua; SUN Senggang; CHEN Xiaowu


    To systematically evaluate the importance of protein synthesis in ischemic preconditioning (PC)-induced ischemic tolerance (IT), temporary middle cerebral artery occlusion (MCAO)by Longa (20 min) was used for PC (ischemic precondioning). Twenty-four hours of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) to compare with non-PC (sham-operated) rats (n=5 for each group). Infarct size and neurological deficits were measured 24 h after PMCAO. Samples of brain were taken for the determination of HSP70 expression by Western blot analysis. The effects of the protein synthesis inhibitor cycloheximide administered just before PC or administered long after PC but just before PMCAO on IT were also determined (n=5 for each group). Our results showed that hemispheric infarct was significantly reduced (P<0.01) only if PC was performed after 24 h, and PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). Cycloheximide eliminated ischemic PC-induced IT effects on both brain injury and neurological deficits if administered before PC but not if administered long after PC but before PMCAO. PC produced no brain injury but did increase HSP70 protein 24 h after PC. Cycloheximide eliminated that effect. The results suggest that PC is a powerful inducer of ischemic brain tolerance as reflected by the preservation of brain tissue and motor function. PC induces IT that is dependent on de novo protein synthesis.

  12. Polydopamine-Encapsulated Fe3O4 with an Adsorbed HSP70 Inhibitor for Improved Photothermal Inactivation of Bacteria. (United States)

    Liu, Dongdong; Ma, Liyi; Liu, Lidong; Wang, Lu; Liu, Yuxin; Jia, Qi; Guo, Quanwei; Zhang, Ge; Zhou, Jing


    Photothermal treatment, a new approach for inactivation of bacteria and pathogens that does not depend on traditional therapeutic approaches, has recently received much attention. In this study, a new type of nanoplatform (PDA@Fe3O4 + PES) was fabricated by using polydopamine (PDA, a photothermal conversion agent) to encapsulate Fe3O4 (a magnetic nanoparticle) and support 2-phenylethynesulfonamide (PES, an inhibitor of heat shock protein 70 (HSP70)). Upon near-infrared light irradiation, the increased temperature weakens π-π and hydrogen bonding interactions, and PES is released from the PDA@Fe3O4 + PES. The released PES inhibits the function of HSP70, reducing bacterial tolerance to photothermal therapy and improving the therapeutic effect against infectious bacterial pathogens. After treatment, PDA@Fe3O4 + PES can be recovered using the magnetic property of the Fe3O4 cores. Consequently, PDA@Fe3O4 + PES possesses the potential to be a recyclable photothermal agent for enhanced photothermal bacterial inactivation without causing secondary pollution.

  13. Cryo-thermal therapy elicits potent anti-tumor immunity by inducing extracellular Hsp70-dependent MDSC differentiation (United States)

    Zhu, Jun; Zhang, Yan; Zhang, Aili; He, Kun; Liu, Ping; Xu, Lisa X.


    Achieving control of metastatic disease is a long-sought goal in cancer therapy. Treatments that encourage a patient’s own immune system are bringing new hopes in reaching such a goal. In clinic, local hyperthermia and cryoablation have been explored to induce anti-tumor immune responses against tumors. We have also developed a novel therapeutic modality of cryo-thermal treatment by alternating liquid nitrogen (LN2) cooling and radio frequency (RF) heating, and better therapeutic effect was achieved in treating metastatic cancer in animal model. In this study, we investigated the mechanism of systemic immune response elicited by cryo-thermal therapy. In the 4T1 murine mammary carcinoma model, we found that local cryo-thermal therapy resulted in a considerable reduction of distant lung metastases, and improved long-term survival. Moreover, results of tumor re-challenge experiments indicated generation of a strong tumor-specific immune memory after the local treatment of primary tumors. Our further study indicated that cryo-thermal therapy caused an elevated extracellular release of Hsp70. Subsequently, Hsp70 induced differentiation of MDSCs into mature DCs, contributing to the relief of MDSCs-mediated immunosuppression and ultimately the activation of strong anti-tumor immune response. Our findings reveal new insight into the mechanism of robust therapeutic effects of cryo-thermal therapy against metastatic cancers.

  14. Differences in body temperature, cell viability, and HSP-70 concentrations between Pelibuey and Suffolk sheep under heat stress. (United States)

    Romero, Rosita Denny; Montero Pardo, Arnulfo; Montaldo, Hugo Horacio; Rodríguez, Ana Delia; Hernández Cerón, Joel


    Pelibuey and Suffolk sheep were compared as to their capacity to regulate body temperature under environmental hyperthermia by measuring their differences in cellular response to heat stress (HS). In a first experiment, seven Pelibuey and seven Suffolk ewes were kept in a climatic chamber for 6 h daily during 10 days (temperatures within the 18 to 39.5 °C range). As chamber temperature rose, sheep rectal temperature increased in both groups, but to a lesser extent in Pelibuey (0.3 °C) than in Suffolk sheep (0.7 °C) (P  0.05]. HS significantly increased HSP-70 average concentrations for both breeds at 43 °C. A significant effect was observed for the breed by temperature interaction (P  0.05). In conclusion, Pelibuey sheep show more effective body temperature regulation under conditions of environmental hyperthermia. Also, cell viability after HS was higher in Pelibuey than in Suffolk, an effect that could be mediated by an HSP-70-related mechanism.

  15. A role for cytosolic hsp70 in yeast [PSI(+)] prion propagation and [PSI(+)] as a cellular stress. (United States)

    Jung, G; Jones, G; Wegrzyn, R D; Masison, D C


    [PSI(+)] is a prion (infectious protein) of Sup35p, a subunit of the Saccharomyces cerevisiae translation termination factor. We isolated a dominant allele, SSA1-21, of a gene encoding an Hsp70 chaperone that impairs [PSI(+)] mitotic stability and weakens allosuppression caused by [PSI(+)]. While [PSI(+)] stability is normal in strains lacking SSA1, SSA2, or both, SSA1-21 strains with a deletion of SSA2 cannot propagate [PSI(+)]. SSA1-21 [PSI(+)] strains are hypersensitive to curing of [PSI(+)] by guanidine-hydrochloride and partially cured of [PSI(+)] by rapid induction of the heat-shock response but not by growth at 37 degrees. The number of inheritable [PSI(+)] particles is significantly reduced in SSA1-21 cells. SSA1-21 effects on [PSI(+)] appear to be independent of Hsp104, another stress-inducible protein chaperone known to be involved in [PSI(+)] propagation. We propose that cytosolic Hsp70 is important for the formation of Sup35p polymers characteristic of [PSI(+)] from preexisting material and that Ssa1-21p both lacks and interferes with this activity. We further demonstrate that the negative effect of heat stress on [PSI(+)] phenotype directly correlates with solubility of Sup35p and find that in wild-type strains the presence of [PSI(+)] causes a stress that elevates basal expression of Hsp104 and SSA1.

  16. 尼美舒利诱导人肝癌SMMC-7721细胞凋亡并下调HSP70基因表达%Downregulation of HSP70 gene expression and apoptosis in human hepatocellular carcinoma SMMC-7721 cells induced by nimesulide in vitro

    Institute of Scientific and Technical Information of China (English)

    殷国志; 涂康生; 韩少山; 王军; 刘青光; 姚英民


    目的:探讨尼美舒利诱导人肝癌SMMC-7721细胞凋亡的作用及其机制.方法:以不同浓度的尼美舒利处理体外培养的人肝癌SMMC-7721细胞,MTT法检测细胞活力,流式细胞术检测细胞凋亡率,RT-PCR和/或Western blot法检测caspase-9和PARP的剪切情况及HSP70的表达水平;通过RNAi技术沉默HSP70基因表达,观察其对细胞凋亡的影响.结果:尼美舒利可抑制人肝癌SMMC-7721细胞生长,诱导细胞凋亡,导致caspase-9和PARP的剪切活化及HSP70的mRNA和蛋白表达下调;在人肝癌SMMC-7721细胞中转染靶向HSP70的siRNA可沉默HSP70表达、剪切活化caspase-9和PARP,进而显著促进细胞凋亡.结论:尼美舒利可诱导人肝癌SMMC7721细胞凋亡,这一作用可能与抑制HSP70表达有关.%AIM: To investigate the effect of nimesulide on cell apoptosis and possible mechanism in human hepatocellular carcinoma SMMC-7721 cells. METHODS; SMMC-7721 cells were treated with nimesulide at different concentrations. Cell viability was assessed by MTT assay. Cell apoptosis rate was determined with flow cytometry. The cleavage activity of PARP and caspase-9 and the expression of HSP70 were evaluated using RT-PCR and Western blotting. The influence of HSP70 on cell apoptosis was observed using RNA interference silencing HSP70 expression. RESULTS: Nimesulide significantly inhibited cell growth in SMMC-7721 cells in a time- and concentration-dependent manner, and induced cell apoptosis in a concentration-dependent manner. Moreover, nimesulide promoted the cleavage of caspase-9 and PARP and inhibited the mRNA and protein expression of HSP70. Through the specific inhibition on HSP70 gene with siRNA, cell apoptosis increased, and the apoptosis was enhanced by the cleavage activity of caspase-9 and PARP. CONCLUSION; Nimesulide could inhibit cell growth and induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells via the downregulation of HSP70.

  17. Developmental and hyperthermia-induced expression of the heat shock proteins HSP60 and HSP70 in tissues of the housefly Musca domestica: an in vitro study

    Directory of Open Access Journals (Sweden)

    Sunita Sharma


    Full Text Available The expression pattern of two major chaperones, the heat shock proteins (HSPs HSP60 and HSP70 was studied in vitro in tissues of the housefly Musca domestica during larval and adult stages of development to identify their immunological relatives and understand their functional significance in normal cellular activities and during thermal stress. Fluorographs of labeled polypeptides and western blots demonstrated that both HSPs are expressed constitutively and heat-induced in all the larval and adult cell types examined. The pattern of whole tissue immunocytochemical staining using anti-HSP60 and anti-HSP70 antibodies corresponded well with the observations from western blots or fluorographs. In developing oocytes, both constitutive and heat inducible expression of HSP60 were regulated in an oocyte stage-specific manner. In unstressed ovaries the expression of these proteins was less pronounced in early stage oocytes (1st - 8th than at later stages (9th and onward. The heat shock, however, induced both HSP70 and HSP60 to a significantly high level in early stage oocytes (1st-8th as compared to their respective controls. Our findings indicate the involvement of the HSP60 and HSP70 proteins in the development, growth and differentiation of both somatic and germ line tissues. Furthermore, the enhanced co-expression of HSP70 and HSP60 upon heat shock in various larval and adult cell types suggests the possible role of HSP60 in thermoprotection.

  18. Quantitative assessment of hsp70, IL-1β and TNF-α in the spinal cord of dogs with E40K SOD1-associated degenerative myelopathy. (United States)

    Lovett, M C; Coates, J R; Shu, Y; Oglesbee, M J; Fenner, W; Moore, S A


    Inflammation is involved in the pathogenesis of many neurodegenerative diseases. Canine degenerative myelopathy (DM) is a progressive adult-onset neurodegenerative disease commonly associated with an E40K missense mutation in the SOD1 gene. DM has many similarities to some familial forms of human amyotrophic lateral sclerosis (ALS) and may serve as an important disease model for therapy development. Pro-inflammatory mediators such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and heat shock protein (hsp) 70 play a role in the pathogenesis of ALS. The focus of the current work was to determine whether an inflammatory phenotype is present in canine DM as defined by IL-1β, TNF-α, and hsp70 responses in cerebrospinal fluid (CSF) and spinal cord tissue. Concentrations of hsp70, IL-1β and TNF-α were below the limits of detection by ELISA in the CSF of both normal and DM-affected dogs. Immunohistochemical staining for hsp70 was significantly increased in ependymal cells lining the spinal cord central canal of DM-affected dogs (P = 0.003). This was not associated with increased IL-1β or TNF-α staining, but was associated with increased CD18 staining in the gray matter of DM-affected dogs. These results suggest that hsp70 in spinal cord tissue is a potential inflammatory signature in canine DM.

  19. Combined microwave irradiation and intraarticular glutamine administration-induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model. (United States)

    Fujita, Shinya; Arai, Yuji; Nakagawa, Shuji; Takahashi, Kenji A; Terauchi, Ryu; Inoue, Atsuo; Tonomura, Hitoshi; Hiraoka, Nobuyuki; Inoue, Hiroaki; Tsuchida, Shinji; Mazda, Osam; Kubo, Toshikazu


    The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real-time RT-PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression.

  20. The tumor-selective over-expression of the human Hsp 70 gene is attributed to the aberrant controls at both initiation and elongation levels of transcription

    Institute of Scientific and Technical Information of China (English)


    The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors, linked to the poor prognosis, being refractory to chemo- and radio-therapies as well as the advanced stage of tumorous lesions in particular. However, both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic. By comparing various upstream segments of the Hsp70gene for each's ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line, we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression. Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene, but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage for targeting the therapeutic gene expression to human tumors.

  1. 香蕉果皮2个HSP70基因片段的克隆及其在热激和冷藏过程中的表达%Cloning and expression analysis of two fragments of HSP70 cDNA in the peel of banana during storage at high and low temperature

    Institute of Scientific and Technical Information of China (English)

    贺立红; 陈建业; 王海蓝; 邝健飞; 陆旺金


    温度逆境胁迫可以诱导热激蛋白的表达,而热激处理能够减轻果实冷害。为了探讨温度逆境对香蕉热激蛋白基因表达的调控、辨析香蕉热激蛋白基因的表达与果实冷害的关系,采用同源序列法分离了香蕉果皮HStVO基因序列一根据已经报道的HStVO基因的氨基酸保守序列设计引物、以香蕉果皮总RNA为模板,用RT—PCR方法克隆香蕉的HSP70cDNA,Northern杂交分析该基因在不同贮藏温度下的表达特征。结果得到2个序列不同的HStVO基因片段,分别命名为Ma—HSP70-1和Ma—HSP70—2,Ma—HSP70-1和肘n—HSP70—2之间的碱基同源性为86.9%,氨基酸同源性为97.1%。Northern杂交的结果表明,38℃短期热激处理诱导了Ma-HSP70—2基因的表达,低温冷藏能使2个基因的表达增强。并初步认为Ma-HSP70可能与香蕉果实耐冷性有关。%Many evidences have indicated that temperature stress can induce heat shock protein expression and heat treatment can alleviate chilling injury of fruits. In the present study, two HSP70 fragments were cloned from banana peel using homology-based method in order to investigate their expressions regulated by temperature stress and the relationship between their expressions and chilling injury. Using PCR de- generate primers designed with reference to the conserved amino acid sequences of known HSP70, total RNA extracted from banana peel was used as templates and HSPTO eDNA fragments were amplified by RT-PCR in order to study their expression characteristics under different environments by Northern hy- bridization. Two different eDNA fragments, named Ma-HSP70-1 and Ma-HSP70-2, were cloned. The homology between two HSP70 is 86.9% at nueleotide acid sequence level and 97.1% at amino acid se- quence level. Using Ma-HSP70-1 and Ma-HSP70-2 as probes, Northern hybridization showed that short-time heat pretreatment (38 ℃ )only induced the expression of Ma-HSP70

  2. 绵羊肺炎支原体Y98株未知基因Hsp70(DnaK)的克隆%Using tail-PCR technology to clone the unkonwn gene Hsp70(DnaK) of Mycoplasma ovipneumoniae Y98

    Institute of Scientific and Technical Information of China (English)

    马春骥; 李敏; 赵东; 王玉炯


    绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)是绵羊传染性胸膜肺炎(Contagious ovine pleuropneumo-nia)的主要致病菌。热休克蛋白Hsp70也叫(DnaK),具有分子伴侣和免疫的作用。本试验以MO Y98基因组为模板,通过比对15种支原体Hsp70基因的序列并设计简并引物,分别利用同源克隆和染色体步移—Tail-PCR技术克隆到了四段Hsp70(DnaK)基因片断并进行了核苷酸序列测定。利用序列拼接软件对克隆的四段序列进行序列拼接,通过ORF Finder软件分析出了MO Y98的Hsp70的开放式阅读框架,预测分析该基因是由1 815bp组成,编码604个氨基酸。本试验于国内外首次克隆到了MO的Hsp70基因序列,为MO的抗原研究以及Hsp70的功能研究等奠定了基础。%Mycoplasma ovipneumonia(MO) is the main pathogen causing contagious ovine pleuropneumonia.In this work,comparing 15 species Hsp70 gene and protein sequence of mycoplasma,the conservative sequences were found and degenerate primers were designed.Then,four fragments of MO Hsp70 gene were acquired by homologous cloning combined with Tail-PCR,chromosome walking.Four fragments of MO Hsp70 were assembled and the whole ORF was found by ORF Finder,which was 1815 bp in length that encoded 604 amino acids.MO Hsp70 was cloned the first time at home and abroad.Our results layed the foundation for the further research of Hsp70 function and MO antigens.

  3. Stress-induced phosphorylation of c-Jun-N-terminal kinases and nuclear translocation of Hsp70 in the Wistar rat hippocampus

    Directory of Open Access Journals (Sweden)

    Adžić M.


    Full Text Available Glucocorticoids are key regulators of the neuroendocrine stress response in the hippocampus. Their action is partly mediated through the subfamily of MAPKs termed c-Jun-N-terminal kinases (JNKs,whose activation correlates with neurodegeneration. The stress response also involves activation of cell protective mechanisms through various heat shock proteins (HSPs that mediate neuroprotection. We followed both JNKs and Hsp70 signals in the cytoplasmic and nuclear compartments of the hippocampus of Wistar male rats exposed to acute, chronic, and combined stress. The activity of JNK1 was decreased in both compartments by all three types of stress, while the activity of cytoplasmic JNK2/3 was elevated in acute and unaltered or lowered in chronic and combined stress. Under all stress conditions, Hsp70 translocation to the nucleus was markedly increased. The results suggest that neurodegenerative signaling of JNKs may be counteracted by increase of nuclear Hsp70,especially under chronic stress.

  4. Survival of parvalbumin-immunoreactive neurons in the gerbil hippocampus following transient forebrain ischemia does not depend on HSP-70 protein induction. (United States)

    Ferrer, I; Soriano, M A; Vidal, A; Planas, A M


    HSP-70 was induced in the gerbil following 20 min of forebrain ischemia. The induction, as revealed with immunohistochemistry, is stronger and longer-lasting in CA3 and dentate gyrus than in CA1. Most neurons in this region, except GABAergic interneurons containing the calcium-binding protein parvalbumin, eventually cease to live as a result of delayed cell death. Double-labeling of inducible HSP-70 and parvalbumin has shown that no co-localization occurs in the hippocampus and neocortex of the gerbil in this model of transient forebrain ischemia. These results show that different thresholds of sensitivity and vulnerability exist for different subpopulations of neurons in the ischemic hippocampus, and suggest that HSP-70 protein induction is probably not essential for the survival of particular neuronal subpopulations subjected to transient ischemia.

  5. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70. (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien


    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.

  6. FLZ, a novel HSP27 and HSP70 inducer, protects SH-SY5Y cells from apoptosis caused by MPP(+). (United States)

    Kong, Xiang-Chen; Zhang, Dan; Qian, Cheng; Liu, Geng-Tao; Bao, Xiu-Qi


    Heat shock proteins (HSPs) play an essential role in various neurodegenerative diseases. Manipulation of upregulation of HSPs in cells has been demonstrated to provide a therapeutic strategy to counteract the misfolding and aggregation of proteins that resulted in neurodegenerative disease. Our previous studies have shown that FLZ, a synthetic novel derivative of squamosamide from a Chinese herb, had potent neuroprotective effect against several experimental Parkinson's disease (PD) models. However, the mechanism of its neuroprotective effect is still not clarified. The present study demonstrated that FLZ induced HSP27 and HSP70 proteins and mRNA expression in a time- and dose-dependent manner in SH-SY5Y cells. Further studies showed that FLZ treatment stimulated the activation of heat shock factor 1 (HSF1) and its regulatory kinase Akt. Inactivation of Akt pathway by the PI3K inhibitor LY294002 blocked the expression of HSP27 and HSP70 induced by FLZ. Moreover, the inducing effects of FLZ on HSP27, HSP70, and HSF1 were all blocked by quercetin, an inhibitor of HSP biosynthesis. The cytoprotective effect of HSP27/HSP70 induced by FLZ against MPP(+) was assessed in SH-SY5Y cells. The pretreatment of FLZ significantly induced the accumulations of HSP27/HSP70 and suppressed the apoptosis caused by MPP(+) in SH-SY5Y cells. However, the protective effects of FLZ against MPP(+) were significantly blocked by quercetin, which indicated that the cytoprotective action of FLZ against MPP(+)-induced apoptosis is at least partially mediated by its induction of HSP27/HSP70. These results provide new evidence for elucidating the mechanism of the neuroprotective effect of FLZ against PD.

  7. Characterization of two heat shock proteins (Hsp70/Hsc70) from grass carp (Ctenopharyngodon idella): evidence for their differential gene expression, protein synthesis and secretion in LPS-challenged peripheral blood lymphocytes. (United States)

    Zhang, Anying; Zhou, Xiaofei; Wang, Xinyan; Zhou, Hong


    Two cDNAs, encoding the stress-inducible 70-kDa heat shock protein (Hsp70) and the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), were isolated from grass carp. The Hsp70 and Hsc70 cDNAs were 2250 bp and 2449 bp in length and contained 1932 bp and 1953 bp open reading frames, respectively. Tissue distribution results showed that Hsp70/Hsc70 was highly expressed in gill, kidney, head kidney and peripheral blood lymphocytes (PBLs). Using grass carp PBLs as a cell model, effects of lipopolysaccharide (LPS) on the mRNA and protein levels of Hsp70/Hsc70 were examined. In this case, LPS increased the mRNA expression of Hsp70 in a time- and dose-dependent manner, but had no effect on Hsc70 mRNA expression. In agreement with this, LPS elevated the intracellular Hsp70 markedly, but not the Hsc70 protein levels in parallel experiments. Furthermore, Hsp70 protein was also detected in culture medium. Moreover, inhibition of LPS on Hsp70 release in a time-dependent manner was observed, indicating that there may be a dynamic balance between Hsp70 stores and Hsp70 release in grass carp PBLs following exposure to LPS. Taken together, these results not only shed new insights into the different regulations of LPS on Hsp70/Hsc70 gene expression, protein synthesis and release, but also provide a basis for further study on the functional role of Hsp70 in fish immune response.

  8. Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction (United States)

    Khan, Imran; Lee, Kyeong-Lim; Fakruzzaman, Md.; Song, Seok-Hwan; Ihsan-ul-Haq; Mirza, Bushra; Yan, Chang Guo; Kong, Il-Keun


    Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro. PMID:26831738

  9. 2-phenylethynesulfonamide Prevents Induction of Pro-inflammatory Factors and Attenuates LPS-induced Liver Injury by Targeting NHE1-Hsp70 Complex in Mice.

    Directory of Open Access Journals (Sweden)

    Chao Huang

    Full Text Available The endotoxin-mediated production of pro-inflammatory cytokines plays an important role in the pathogenesis of liver disorders. Heat shock protein (Hsp70 overexpression has established functions in lipopolysaccharide (LPS-mediated inflammatory response. However, little is known about the role of Hsp70 activity in LPS signaling. We hypothesized that inhibition of Hsp70 substrate binding activity can ameliorate LPS-induced liver injury by decreasing induction of pro-inflammatory factors. In this study, C57/BL6 mice were injected intraperitoneally with LPS and 2-phenylethynesulfonamide (PES, an inhibitor of Hsp70 substrate binding activity. We found that i. PES prevented LPS-induced increase in serum alanine aminotransferase (ALT and aspartate aminotransferase (AST activity, infiltration of inflammatory cells, and liver cell apoptosis; ii. PES reduced inducible nitric oxide synthase (iNOS protein expression as well as serum nitric oxide (NO, tumor necrosis factor-α (TNF-α, and interleukin-6 (IL-6 content in LPS-stimulated mice; iii. PES reduced the mRNA level of iNOS, TNF-α, and IL-6 in LPS-stimulated liver. iiii. PES attenuated the degradation of inhibitor of κB-α (IκB-α as well as the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB in LPS-stimulated liver. Similar changes in the protein expression of inflammatory markers, IκB-α degradation, and NF-κB phosphorylation and nuclear translocation were observed in RAW 264.7 cells. Further mechanistic studies revealed that PES remarkably reduced the elevation of [Ca(2+]i and intracellular pH value (pHi in LPS-stimulated RAW 264.7 cells. Furthermore, PES significantly reduced the increase in Na(+/H(+ exchanger 1 (NHE1 association to Hsp70 in LPS-stimulated macrophages and liver, suggesting that NHE1-Hsp70 interaction is required for the involvement of NHE1 in the inflammation response. In conclusion, inhibition of Hsp70 substrate binding activity in vivo reduces the

  10. 旋毛虫重组HSP70对小鼠巨噬细胞TLR2/4表达的影响%Effect of 70 ku recombinant HSP70 from Trichinella spiralis on expres-sion of TLR2/4 in murine macrophage

    Institute of Scientific and Technical Information of China (English)

    李成; 禹洋; 徐佳; 刘畅; 周兴东; 李晓云


    观察旋毛虫重组热休克蛋白HSP70(rTs-HSP70)对体外培养小鼠巨噬细胞Toll样受体TLR2/4表达的影响。体外常规培养小鼠巨噬细胞系RAW264.7,分别加入不同浓度rTs-HSP70刺激培养24 h,半定量PCR方法检测培养细胞TLR2/4 mRNA表达,流式细胞仪检测表面标志分子CD80和CD86表达。平行设立不加刺激物阴性对照组,细菌脂多糖LPS阳性对照组;同时试验比较5μg·mL-1浓度的HSP70组和HSP70+LPS组(HSP70预刺激后再加入LPS刺激培养12 h)RAW264.7细胞TLR2/4 mRNA表达差异。结果表明,低浓度rTs-HSP70与巨噬细胞活化呈正相关,浓度为5μg·mL-1时,TLR2/4表达水平达到峰值(P5μg·mL-1)则对巨噬细胞RAW264.7表现为抑制作用;当对RAW264.7进行旋毛虫HSP70预刺激,可引起免疫抑制效应,抑制LPS对巨噬细胞TLR2/4的活化反应。试验可为旋毛虫相关分子免疫调节机制研究及旋毛虫病防治提供参考。%To observe the effect of 70 kDa recombinant heat shock protein from Trichinella spiralis (rTs-HSP70) on expression of TLR2/4 (toll-like receptors) in murine macrophage in vitro. Murine macrophage RAW264.7 cell lines were incubated in twelve-well plates and different concentrations of rTs-HSP70 were added, co-culturing for 24 hours. Then the cells’TLR2/4 expression were tested by semi-quantitative PCR, those of surface markers CD80 and CD86 by flow cytometry. Cells receiving no stimulation were taken as negative control, bacterial lipopolysaccharide (LPS) as positive control. At the same time, comparative studies on incubation with rTs-HSP70 alone, rTs-HSP70 in combination with LPS, were conducted at concentration 5 µg·mL-1 of HSP70. The results indicated that lower concentra-tions of rTs-HSP70 had a positive correlation with macrophages activation, TLR2/4 expression peaked at concentration of 5 µg·mL-1 (P5 µg·mL-1), a inhibitory effect on macrophage could be observed. Pre-stimulation of the

  11. Expression and significance of HSP70 in conge nital ca taract lens ep ithelial cells of mice%HSP70在先天性白内障小鼠晶状体上皮细胞中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    王萍; 蔡小军; 朱怡; 熊期; 张菁; 李朝辉; 余爱华


    目的:探讨热休克蛋白(heat shock protein, HSP)70在先天性白内障小鼠模型晶状体上皮细胞中的表达及意义。方法先天性白内障 BALB/c -Cat 小鼠为实验组,正常BALB/c 小鼠为对照组,每组16只。将实验组与对照组均各取8只分别进行 Western blot 蛋白印迹法和逆转录聚合酶链反应(RT -PCR)法检测小鼠眼睛晶状体上皮细胞中 HSP70HSP70基因的表达水平,以β-actin 作为内参校正,比较2组间 HSP70表达的差异。结果HSP70在小鼠晶状体上皮细胞的相对表达值实验组为0.1050±0.0414,对照组为0.1887±0.0236,2组比较差异有统计学意义(t =4.9720,P <0.01);小鼠晶状体上皮细胞 HSP70基因的相对表达值实验组为0.1313±0.0035,对照组为0.334±0.0042,2组比较差异有统计学意义(t =37.026,P <0.01)。结论HSP70在先天性白内障小鼠病情的发生、发展中能起一定的细胞保护作用。%Objective It is to approach the expression of heat shock protein 70 (HSP70) in lens epithelial cells (LECs) in mice model of congenital cataract and clarify the effect of HSP 70 on pathogenesis of congenital cataract .Methods The lens capsule membrane in mice was chose as the research object , there was 16 cases (32 eyes) of normal BALB /c mouse in control group, and 16 cases (32 eyes) of congenital cataract BALB /c -Cat mouse in experimental group .The expression of HSP70 protein and HSP 70 gene was assayed using Western blot method of protein imprinting and reverse transcription polymerase chain reaction (RT -PCR) method in the two groups of mice eye lens epithelial cells .Using β-actin as the confidential ref-erence items for correction, the differences of HSP70 expression was compared between the two groups .Results The relative values of HSP70 expression to β-actin is 0.105 0 +0.041 4 in congenital cataract LECs , while is 0.188 7 +0.023 6 in nor-mal control

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

    Directory of Open Access Journals (Sweden)

    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

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

    Institute of Scientific and Technical Information of China (English)

    井然; 钱捷; 弓景波; 梅竹松; 王新兴; 钱令嘉


    group,significant increase of rectal temperature in rats was observed in heat stress groups(36, 37, 38, 39 ℃)(P<0.05). Q-T interval prolongation and R wave amplitude descend were observed in electrocardiogram in heat stress groups (37, 38, 39 ℃), and the rate of abnormal electrocardiogram in heat stress groups (36, 37, 38, 39 ℃) also increased as the ambient temperature increased(P<0.05). When environmental temperature was over 38 ℃, the activity of LDH and CK-MB in serum in heat stress groups increased significantly (P<0.05). In 36 ℃. and 37 ℃ heat stress groups, HSP70 level increased remarkably (P<0.05) as ambient temperature increased. But when ambient temperature was over 38 ℃ , HSP70 expression decreased with increased temperature,while still significantly higher than the normal temperature group. Conclusion Heat stress may induce the myocardium damage and change of HSP70 expression in blood lymphocytes in rats, which suggests that the HSP70 in blood lymphocytes maybe is a molecular marker and HSP70 plays a role in myocardium injury induced by heat exposure.

  14. 艾灸及槲皮素预处理对胃黏膜细胞凋亡及HSP70表达的影响%The Effect of Quercetin Blocking HSP70 Expressing on Pre-Moxibustion Suppressing Gastric Mucosal Cell Apoptos

    Institute of Scientific and Technical Information of China (English)

    易受乡; 郁洁; 常小荣; 彭艳; 林亚平



  15. Studies on effect of different seasons on expression of HSP70 and HSP90 gene in sperm of Tharparkar bull semen

    Directory of Open Access Journals (Sweden)

    J.S. Rajoriya


    Conclusion: It was concluded from the present study that there was no significant difference in the mRNA expression of HSP 70 and HSP 90 between the winter and summer season, presence of similar type of stress resistant spermatozoa in Tharparkar bull semen and the semen can be cryopreserved throughout the year in this prestigious Indian breed.

  16. Different immunohistochemical levels of Hsp60 and Hsp70 in a subset of brain tumors and putative role of Hsp60 in neuroepithelial tumorigenesis

    Directory of Open Access Journals (Sweden)

    F. Rappa


    Full Text Available In this work we analysed, by immunohistochemistry, a series of brain tumors to detect the levels and cellular distribution of Hsp60 and Hsp70. We found that Hsp60 levels were significantly higher than those of Hsp70 in neuroepithelial tumors, while levels of both molecules were not significantly different from each other in meningeal neoplasms. In particular, Hsp60 immunopositivity was present mainly at the cytoplasmic level, while Hsp70 immunopositivity was found both in the cytoplasm and in the nucleus of tumor cells. The levels of these molecules in healthy control cells were always very low. Finally, Hsp60 and Hsp70 levels did not correlate with the different types (WHO grade of neoplasm. Our results are partially in agreement with previous studies and suggest that Hsp60 is not increased by a passive phenomenon (e.g., due to the stress caused by the peritumor environment on cancer cells but may be actively implicated in tumor progression, e.g. inhibiting tumor cell death or antitumor immune system response, as already postulated in vitro. We also briefly discuss the most recent publications on the extramitochondrial localization of Hsp60 in tumor cells and its role in tumor progression.

  17. Fisetin, a dietary flavonoid, induces apoptosis of cancer cells by inhibiting HSF1 activity through blocking its binding to the hsp70 promoter. (United States)

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


    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.

  18. Monitoring the change of mitochondrial morphology and its metabolism of the breast cancer cells with the treatment of Hsp70 inhibitor during heat shock by fluorescence imaging (United States)

    Yu, Biying; Yang, Hongqin; Zhang, Xiaoman; Li, Hui


    Heat shock (HS) is one of the best-studied exogenous cellular stresses, and all cellular compartments and metabolic processes are involved in HS response. The heat shock proteins (Hsps) expression enhanced during HS mainly localized in subcellular compartments, such as cytosol, endoplasmic reticulum and mitochandria. The major inducible heat shock protein 70 (Hsp70) modulate cellular homeostasis and promote cellular survival by blocking a caspase independent cell death through its association with apoptosis inducing factor. Mitochondria as the critical elements of HS response that participate in key metabolic reactions, and the changes in mitochonrial morphology may impact on mitochondrial metabolism. In this paper, the changes of mitorchondrial morphology in breast cancer cell have been monitored in real time after heat shock (43 °) by the fluorescence imaging, and the influence of Hsp70 inhibitor on mitochandrial structures have also been investigated. Then the information of mitochondrial metabolism which can be characterized by the level of the mitochondrial membrane potential has also been obtained wihout/with the treatment of Hsp70 inhibitor. Our data indicated that the mitochandrial morphology were related with the mitochandrial membrane potential, and the mitochandrial membrane potential was influenced significantly with the treatment of Hsp70 inhibitor during HS.

  19. Effects of covering behavior and exposure to a predatory crab Charybdis japonica on survival and HSP70 expression of juvenile sea urchins Strongylocentrotus intermedius. (United States)

    Zhao, Chong; Ji, Nanjing; Zhang, Binglong; Sun, Ping; Feng, Wenping; Wei, Jing; Chang, Yaqing


    Predation is a complex process among predator, prey and environment. Juvenile sea urchins are more susceptible to predators than adults, which affects community structure. Behavior is involved in anti-predator responses by changes in the expression of anti-predator responsive genes. Here, we investigated the effects of exposure to a predatory crab Charybdis japonica and covering behavior on survival and HSP70 expression of juvenile sea urchins Strongylocentrotus intermedius. C. japonica consumed large numbers of juvenile S. intermedius in 12 hours with a mortality of 34.17±11.43%. Covering behavior did not significantly reduce predation. Exposure to C. japonica did not significantly upregulate HSP70 expression of juvenile S. intermedius in 12 hours. Covering behavior showed no significant regulative effect on the gene expression of HSP70 of juvenile S. intermedius exposed to C. japonica for 12 hours. The results indicate that the anti-predator function of covering behavior is limited and that HSP70 expression does not appear to play an important role in the anti-predator process of S. intermedius.

  20. 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.


    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 tr


    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...

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

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


    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.


    Institute of Scientific and Technical Information of China (English)

    PENG Na; CHANG Xiao-rong; YI Shou-xiang; PENG Yan; YAN Jie


    Objective: To observe effect of moxibustion at Zusanli (足三里 ST 36) and Liangmen (梁门 ST 21 ) on expression of heat shock protein 70 (HSP70) in gastric mucosa of the rat of stress ulcer (SU) to explore the mechanism of moxibustion in resisting oxidative injury of the gastric mucosa. Methods: Sixty SD rats were evenly randomized into 4 groups, a blank group, a model group, an acupoint moxibustion group and a non-acupoint moxibustion group. Water restraint stress (WRS) method was used to make stress gastric ulcer rat model. The ulcerative index (UI) of gastric mucosa was evaluated by using GUTH method, the gastric mucosa blood flux (GMBF) was detected by a laser Doppler bloodflow monitor, and HSP70 expression and malondialdehyde (MDA) content in the gastric mucosa were determined respectively with immunohistochemical and thiobarbiturate methods. Results: Moxibustion at Zusanli (ST 36) and Liangmen (ST 21 ) significantly decreased UI, up-regulated HSP70 expression, increased GMBF, and decreased MDA content in the gastric mucosa in the rat of stress gastric ulcer, with significant differences as compared with the model group and the non-acupoint moxibustion group ( P < 0.05 or P < 0.01 ). Conclusion: Moxibustion at Zusanli (ST 36) and Liangmen (ST 21 ) can induce high expression of HSP70 and decrease MDA content in the gastric mucosa, so as to resist oxidative injury, with relative acupoint specificity.

  4. Tyrosol prevents ischemia/reperfusion-induced cardiac injury in H9c2 cells: involvement of ROS, Hsp70, JNK and ERK, and apoptosis. (United States)

    Sun, Liwei; Fan, Hang; Yang, Lingguang; Shi, Lingling; Liu, Yujun


    Ischemia-Reperfusion (I/R) injury causes ROS overproduction, creating oxidative stress, and can trigger myocyte death, resulting in heart failure. Tyrosol is an antioxidant abounded in diets and medicine. Our objective was to investigate the protective effect of tyrosol on I/R-caused mortality in H9c2 cardiomyocytes through its influence on ROS, Hsp70, ERK, JNK, Bcl-2, Bax and caspase-8. A simulated I/R model was used, myocytes loss was examined by MTT, and ROS levels were measured using DCFH-DA. Nuclear condensation and caspase-3 activity were assessed by DAPI staining and fluorometric assay. Phosphorylated ERK and JNK were determined by electrochemiluminescent ELISA, and Hsp70, Bcl-2, Bax and caspase-8 were examined by Western blotting. Results show that tyrosol salvaged myocyte loss, inhibited nuclear condensation and caspase-3 activity dose-dependently, indicating its protection against I/R-caused myocyte loss. Furthermore, tyrosol significantly inhibited ROS accumulation and activation of ERK and JNK, augmenting Hsp70 expression. Besides, tyrosol inhibited I/R-induced apoptosis, associated with retained anti-apoptotic Bcl-2 protein, and attenuated pro-apoptotic Bax protein, resulting in a preservation of Bcl-2/Bax ratio. Finally, tyrosol notably decreased cleaved caspase-8 levels. In conclusion, cytoprotection of tyrosol in I/R-caused myocyte mortality was involved with the mitigation of ROS, prohibition of the activation of ERK, JNK and caspase-8, and elevation of Hsp70 and Bcl-2/Bax ratio.

  5. Tyrosol Prevents Ischemia/Reperfusion-Induced Cardiac Injury in H9c2 Cells: Involvement of ROS, Hsp70, JNK and ERK, and Apoptosis

    Directory of Open Access Journals (Sweden)

    Liwei Sun


    Full Text Available Ischemia-Reperfusion (I/R injury causes ROS overproduction, creating oxidative stress, and can trigger myocyte death, resulting in heart failure. Tyrosol is an antioxidant abounded in diets and medicine. Our objective was to investigate the protective effect of tyrosol on I/R-caused mortality in H9c2 cardiomyocytes through its influence on ROS, Hsp70, ERK, JNK, Bcl-2, Bax and caspase-8. A simulated I/R model was used, myocytes loss was examined by MTT, and ROS levels were measured using DCFH-DA. Nuclear condensation and caspase-3 activity were assessed by DAPI staining and fluorometric assay. Phosphorylated ERK and JNK were determined by electrochemiluminescent ELISA, and Hsp70, Bcl-2, Bax and caspase-8 were examined by Western blotting. Results show that tyrosol salvaged myocyte loss, inhibited nuclear condensation and caspase-3 activity dose-dependently, indicating its protection against I/R-caused myocyte loss. Furthermore, tyrosol significantly inhibited ROS accumulation and activation of ERK and JNK, augmenting Hsp70 expression. Besides, tyrosol inhibited I/R-induced apoptosis, associated with retained anti-apoptotic Bcl-2 protein, and attenuated pro-apoptotic Bax protein, resulting in a preservation of Bcl-2/Bax ratio. Finally, tyrosol notably decreased cleaved caspase-8 levels. In conclusion, cytoprotection of tyrosol in I/R-caused myocyte mortality was involved with the mitigation of ROS, prohibition of the activation of ERK, JNK and caspase-8, and elevation of Hsp70 and Bcl-2/Bax ratio.

  6. Expressions of HSP 70 and NF-κB in the peripheral blood lymphocyte of chronic gastritis patients of different syndrome patterns

    Institute of Scientific and Technical Information of China (English)



    Objective To study the expressions of heat shock protein 70 (HSP 70) and nuclear factor-kappa B(NF-κB) in the peripheral blood lymphocyte of chronic gastritis (CG) patients of Pi-Wei hygropyrexia syndrome (PWHS) and Pi-qi deficiency syndrome(PQDS),and to explore their correlation with Helicobacter pylori(Hp) infection. Methods

  7. The Changes of HSP70 in Skeletal Muscle after an Exhaustive Eccentric Exercise and the Effects of Acupuncture%一次力竭离心运动后大鼠骨骼肌HSP70的变化及针刺对其影响

    Institute of Scientific and Technical Information of China (English)

    李俊平; 马延超; 张炜


    Purpose:The purpose of this study was to observe the changes of HSP70 expression in rat gastrocnemius muscle after an exhaustive eccentric exercise,and to explore the effects of acupuncture on HSP70 expression in skeletal muscle after exercise.Method:Male SD rats were randomly divided into four groups: control group,non-acupuncture group and acupuncture group after an exhaustive eccentric exercise.The rats of non-acupuncture and acupuncture group were carried out an exhaustive eccentric exercise.After the exercise,the rats of acupuncture group were handled acupuncture on the gastrocnemius vertically from the further side(the angle of the needle was 30 degree) and through the belly of gastrocnemius where the needle stayed for 5 minutes.Then the gastrocnemius was obtained from rats of different groups.The expression of HSP70 of gastrocnemius was detected by western blot and immunofluorescence histochemical method.Results:1)The expression of HSP70 of gastrocnemius in non-acupuncture group were increased markedly immediately,12 hours and 24 hours after exhaustive eccentric exercise(P0.05).2)The expression of HSP70 of gastrocnemius in acupuncture group were increased markedly only 12 hours after exhaustive eccentric exercise(P0.05).3)The expression of HSP70 of gastrocnemius in acupuncture group was significantly lower than non-acupuncture group immediately and 24 hours after exhaustive eccentric exercise(P0.05).4)The expression of HSP70 of gastrocnemius in non-acupuncture group mainly located in cytoplasm.The expression of HSP70 of gastrocnemius in acupuncture group mainly located in nucleus immediately after exhaustive eccentric exercise,and then transferred to cytoplasm gradually.Conclusions:1)Immediately after an exhaustive eccentric exercise,HSP70 expression of rat gastrocnemius were induced markedly,and then decreased gradually.2) HSP70 expression of rat gastrocnemius induced by an exhaustive eccentric exercise might be

  8. A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter* (United States)

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H.; Freissmuth, Michael; Sucic, Sonja


    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay. PMID:25202009

  9. HSP70在乐果染毒U87细胞中的保护作用%Protective Effect of HSP70 in U87 Cell Line Treated with Dimethoate

    Institute of Scientific and Technical Information of China (English)

    谢芳; 吴同俊; 吴强恩; 周志俊


    目的研究HSP70在乐果染毒U87细胞中的保护作用.方法预先给予细胞43℃热应激1 h,然后以浓度为100μmol/L的乐果对细胞分别进行4、8、16h染毒,染毒结束测AChE和SOD活性并与未经热应激诱导组比较.结果在不同染毒时间,预先经热应激处理的试验组,其AChE及SOD活性都较直接染毒的对照组高,差异有非常显著性(P<0.01),酶活性的变化率在不同时间组间的差异也具有非常显著性(P<0.01).结论预先进行热应激诱导对乐果染毒U87细胞具有保护作用.

  10. Cloning and phylogenetic analysis of HSP70 gene from cattle Theileria sergenti%牛瑟氏泰勒虫HSP70 cDNA片段的克隆与系统发育分析

    Institute of Scientific and Technical Information of China (English)

    曹世诺; 于龙政; 薛书江; 贾立军; 张守发


    从血液涂片检查为牛瑟氏泰勒虫阳性的病牛血液中提取总BNA.通过RT-PCR技术扩增出牛瑟氏泰勒虫HSP70基因,将其重组到pMD-18T Simple载体后进行克隆、序列测定及分析.结果表明该片段长1966 bp,编码620个氨基酸残基,将该基因片段序列与GenBank中13种已知梨形虫的相应序列进行比较分析,牛瑟氏泰勒虫吉林分离株与已报道的牛瑟氏泰勒虫亲缘关系最近,其次是环形泰勒虫,与马巴贝斯虫亲缘关系较远.

  11. Identification of several cytoplasmic HSP70 genes from the Mediterranean mussel (Mytilus galloprovincialis) and their long-term evolution in Mollusca and Metazoa. (United States)

    Kourtidis, Antonis; Drosopoulou, Elena; Nikolaidis, Nikolas; Hatzi, Vasiliki I; Chintiroglou, Chariton C; Scouras, Zacharias G


    The HSP70 protein family consists one of the most conserved and important systems for cellular homeostasis under both stress and physiological conditions. The genes of this family are poorly studied in Mollusca, which is the second largest metazoan phylum. To study these genes in Mollusca, we have isolated and identified five HSP70 genes from Mytilus galloprovincialis (Mediterranean mussel) and investigated their short-term evolution within Mollusca and their long-term evolution within Metazoa. Both sequence and phylogenetic analyses suggested that the isolated genes belong to the cytoplasmic (CYT) group of the HSP70 genes. Two of these genes probably represent cognates, whereas the remaining probably represent heat-inducible genes. Phylogenetic analysis including several molluscan CYT HSP70s reveals that the cognate genes in two species have very similar sequences and form intraspecies phylogenetic clades, differently from most metazoan cognate genes studied thus far, implying either recent gene duplications or concerted evolution. The M. galloprovincialis heat-inducible genes show intraspecies phylogenetic clustering, which in combination with the higher amino acid than nucleotide identity suggests that both gene conversion and purifying selection should be responsible for their sequence homogenization. Phylogenetic analysis including several metazoan HSP70s suggests that at least two types of CYT genes were present in the common ancestor of vertebrates and invertebrates, the first giving birth to the heat-inducible genes of invertebrates, whereas the other to both the heat-inducible genes of vertebrates and the cognate genes of all metazoans. These analyses also suggest that inducible and cognate genes seem to undergo divergent evolution.

  12. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.

    Directory of Open Access Journals (Sweden)

    Luiz Felipe M Prota

    Full Text Available BACKGROUND: Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR. Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression. METHODS AND RESULTS: Dose-response (1 nM to 10 µM and time course (3 to 48 h curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90 binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70 binding decreased 0.30 fold in an immunoprecipitation assay. CONCLUSION: Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.

  13. 实时荧光定量PCR检测球孢白僵菌热休克蛋白基因hsp70在几种胁迫条件下的表达%Expression analysis of hsp70 gene from Beauveria bassiana under several stress conditions by Realtime-PCR

    Institute of Scientific and Technical Information of China (English)

    谢翎; 陈红梅; 汤强; 蒲顺昌; 李增智; 黄勃


    本研究运用Realtime-PCR技术,对球孢白僵菌Beauveria bassiana hsp70基因在不同胁迫条件下的表达情况进行了检测.从mRNA转录水平探讨了不同胁迫条件对球孢白僵菌hsp70基因表达的影响.结果表明:38℃高温胁迫下,30min时表达量达到最高峰,为对照样品的10.18倍.随后表达量开始下降,至180min时,其表达量降为最低,为对照样品的2.85倍;4℃低温胁迫下,2h检测到hsp70的表达量下降至最低点,为对照样品的0.25倍.随后表达量开始回升,至10h表达量始终维持在对照样品的1.4-1.5倍左右;紫外胁迫下(波长253.7nm),3min后hsp70的表达量快速上升至最高峰,为对照样品的2.33倍.随后表达量迅速下降,至60min表达量始终维持在对照样品的0.2倍左右.因此推测,hsp70基因在球孢白僵菌抵抗高温、低温和紫外二种胁迫方面都可能具有重要作用.同时研究结果也表明,球孢白僵菌hsp70基因启动子在逆境下可引导基因高效表达,因而在抗逆工程菌株构建方面可能具有重要的应用价值.%The expression of hsp70 gene from Beauveria bassiana under several stress conditions was detected using Realtime-PCR, and the effects of different stress conditions on expression of hsp70 gene on mRNA level were explored. The results showed that under high temperature stress (38℃), the expression level of hsp70 gene reached the maximum (10.18 times of the control) in 30 minutes, and then decreased gradually. It reached the minimum (2.85 times of the control) in 180 minutes. Under low temperature stress (4℃), the expression level of hsp70 gene reached the minimum (0.25 times of the control) in 2 hours, and then increased gradually. It keep stable (1.4-1.5 times of the control) in l0 hours. Under UV stress (253.7nm wavelength), the expression level of hsp70 gene reached the maximum (2.33 times of the control) in 3 minutes, and then decreased rapidly. It keep stable (0.2 times of the control) in 60

  14. pHSP70P-EGFP真核表达载体的构建及其在人Chang's肝细胞中的表达%Construction of eukaryotic expression vector pHSP70P-EGFP and its expression in human Chang's liver cell

    Institute of Scientific and Technical Information of China (English)

    高峰; 陈亚军; 杨学文


    目的 构建HSP70启动子与绿色荧光蛋白重组真核表达载体,并在人Chang's肝细胞表达,建立一种新的体外药物肝毒性早期预测细胞模型.方法 提取人Chang's肝细胞基因组DNA,钓取HSP70启动子基因,构建重组载体,经脂质体转染人Chang's肝细胞,用G418筛选稳定转染细胞,用荧光显微镜观察绿色荧光表达情况,用实时荧光定量PCR检测EGFP基因表达量的变化.结果 成功钓取HSP70启动子基因并导入真核表达载体pEGFP-N1;G418以300 ng/mL的终浓度筛选出稳定转染的单克隆细胞;荧光显微镜观察到肝毒性药物酮康唑刺激人Chang's 肝细胞后,可诱导HSP70启动子调控增强型绿色荧光蛋白发出绿色荧光,药物刺激组EGFP mRNA相对表达量与正常对照组比较有统计学意义(t=-14.21,P<0.05).结论 成功构建HSP70启动子与绿色荧光蛋白共表达真核表达载体,获得稳定转染单克隆细胞,并证实HSP70在肝毒性药物刺激下的应激表达,为建立新的体外药物肝毒性早期预测细胞模型进行高通量筛选新药奠定了基础.%Objective To construct the eukaryotic expression vector of HSP70 promoter and green fluorescent protein and expression the recombine proteins in human Chang's liver cells in order to establish a new drugs hepatotoxicity cell model in vitro for early prediction. Method The genome DNA of human Chang's liver cell was extracted and the promoter of HSP70 gene was obtained. HSP70 promoter gene was transfected to human Chang's liver cell by means of pEGFP-Nl vector with liposome. The steadily transfected cells were screened by G418 selection. The expression of green fluorescent protein was observed by fluorescence microscope. The change of the expression of EGFP gene were observed by real - time fluorescence quantitative PCR. Results pEGFP-Nl vector expressing recombi-nant HSP70 promoter and green fluorescent protein was successfully constructed and the steadily transfected

  15. 子痫前期患者胎盘组织中HSP70和NF-κB的表达及意义%Expression of HSP70 and NF-κB in the placental trophoblasts of preg-nant women complicated with preeclampsia

    Institute of Scientific and Technical Information of China (English)

    林培红; 胡继芬


    Objective To investigate the difference and significance between the expression of HSP70 and NF-κB in the placenta tissues of normal pregnancy and preeclampsia patients, in order to find out the relation between HSP70 and NF-κB. Methods From December 2013 to January 2015, 65 pregnant patients in the First Affiliated Hospital of Fujian Medical University were selected. There were 25 healthy pregnant women (normal pregnant group), 17 mild preeclampsia patients (mild preeclampsia group) and 23 severe preeclampsia patients (severe preeclampsia group). The expressions of HSP70 and NF-κB in the placental tissues were measured using immunohistochemistry methods and re-al-time fluorescence quantitative PCR technique. Results HSP70 and NF-κB immunohistochemical results suggested that the staining degree of the normal pregnant group, the mild and severe preeclampsia group increased gradually. The differences of the expressions of HSP70 and NF-κB between the mild, severe preeclampsia group and the normal preg-nant group were significant (P 0.05); the results of HSP70 and NF-κB real-time fluorescence quantitative PCR suggested that, the level of the expression increased in mild preeclampsia group and severe preeclampsia group, as compared with the normal pregnant group, the difference between the mild preeclampsia group, severe preeclampsia group and the normal pregnant group was statistically significant (P0.05);实时荧光定量PCR结果提示:与正常妊娠组比较, HSP70及NF-κB在轻度子痫前期组及轻度子痫前期组中的表达水平增高, 轻度子痫前期组及重度子痫前期组与正常妊娠组两两比较差异有高度统计学意义(P<0.01).相关性分析结果显示,HSP70与NF-κB呈正相关(r=0.692, P<0.01). 结论 子痫前期患者胎盘组织中HSP70和NF-κB表达增加,可能与子痫前期的发生、发展有关.

  16. Hyperthermia classic article commentary: 'Re-induction of hsp70 synthesis: an assay for thermotolerance' by Gloria C. Li and Johnson Y. Mak, International Journal of Hyperthermia 1989;5:389-403. (United States)

    Li, Gloria C; Calderwood, Stuart K


    Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models. We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. These data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia.

  17. Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector.

    Directory of Open Access Journals (Sweden)

    Linfeng Cheng

    Full Text Available Heat shock proteins (HSPs display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV glycoprotein (GP and nucleocapsid protein (NP immunogenicity by heat shock protein 70 (HSP70, a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359-610 aa, HSP70C to the Gn and 0.7 kb fragment of the NP (aa1-274-S0.7. C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7 and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.

  18. Morphological and Molecular Description of Phytophthora insolita Isolated from Citrus Orchard in India

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    Swapnil Bawage


    Full Text Available Citrus, an important cash crop in India, is adversely affected by Phytophthora nicotianae, P. palmivora, and P. citrophthora. Phytophthora insolita is known to be associated with citrus and reported for the first time in India. It is a rare and poorly characterized Phytophthora species, as its natural host and pathogenic impact are unclear. Previously, it was reported only in Taiwan and China; so to confirm our suspected isolate is P. insolita, regions of internal transcribed spacer, elongation factor, beta-tubulin, and cytochrome oxidase genes were sequenced. This study provides description of the lone Indian P. insolita isolate with respect to molecular identity, morphology, mating behaviour, and pathogenicity.

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

    LENUS (Irish Health Repository)

    Walsh, Naomi


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

  20. Insulin Promotes Survival of Amyloid-Beta Oligomers Neuroblastoma Damaged Cells via Caspase 9 Inhibition and Hsp70 Upregulation

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    M. Di Carlo


    Full Text Available Alzheimer's disease (AD and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

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

    Walsh, Naomi; Larkin, AnneMarie; Swan, Niall; Conlon, Kevin; Dowling, Paul; McDermott, Ray; Clynes, Martin


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

  2. Carboxyl terminus of Hsp70-interacting protein (CHIP) is required to modulate cardiac hypertrophy and attenuate autophagy during exercise. (United States)

    Willis, Monte S; Min, Jin-Na; Wang, Shaobin; McDonough, Holly; Lockyer, Pamela; Wadosky, Kristine M; Patterson, Cam


    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a ubiquitin ligase/cochaperone critical for the maintenance of cardiac function. Mice lacking CHIP (CHIP-/-) suffer decreased survival, enhanced myocardial injury and increased arrhythmias compared with wild-type controls following challenge with cardiac ischaemia reperfusion injury. Recent evidence implicates a role for CHIP in chaperone-assisted selective autophagy, a process that is associated with exercise-induced cardioprotection. To determine whether CHIP is involved in cardiac autophagy, we challenged CHIP-/- mice with voluntary exercise. CHIP-/- mice respond to exercise with an enhanced autophagic response that is associated with an exaggerated cardiac hypertrophy phenotype. No impairment of function was identified in the CHIP-/- mice by serial echocardiography over the 5 weeks of running, indicating that the cardiac hypertrophy was physiologic not pathologic in nature. It was further determined that CHIP plays a role in inhibiting Akt signalling and autophagy determined by autophagic flux in cardiomyocytes and in the intact heart. Taken together, cardiac CHIP appears to play a role in regulating autophagy during the development of cardiac hypertrophy, possibly by its role in supporting Akt signalling, induced by voluntary running in vivo.

  3. A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase. (United States)

    Germaniuk, Aleksandra; Liberek, Krzysztof; Marszalek, Jaroslaw


    Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.

  4. Caracterización in silico de las proteínas del choque térmico Hsp70 y Hsp90 deBemisia tabaci (Hemiptera: Aleyrodidae y su posible actividad adaptativa

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    Eneida Torres Cabra


    Full Text Available La mosca blanca, Bemisia tabaci (Hemiptera: Aleyrodidae es una de las plagas más destructivas e invasivas en el mundo, ataca una gran cantidad de cultivos. Se adapta fácilmente a plantas hospederas y a nuevas regiones geográficas, lo que sugiere el desarrollo de mecanismos de control a daños producidos por factores estresantes. Las proteínas Hsp se expresanen los organismos como mecanismo de defensa, actúan como chaperonas en el correcto ensamblaje de las proteínas. En este estudio se realizó una caracterizaciónin silico de las proteínas Hsp70 y Hsp90 de B. tabaci, secuencias obtenidas de NCBI. La determinaciónde los perfiles de hidrofobicidad, polaridad, accesibilidady flexibilidad se obtuvieron con “ProScale” de ExPASy, el perfil de antigenicidad con JaMBW. La secuencia aminoacídica se analizó con GOR IV y SOPMA y la composición de aminoácidos con ProtParam. Para analizar el peso molecular, índice deinestabilidad, índice alifático y gradiente hidropático,con GRAVY. La estructura terciaria se obtuvo con HHpred, y ESyPred3D. Para validar las estructuras 3D se utilizó Procheck, What_check y errat. Hsp70 y Hsp90 de B. tabaci presentan valores bajos de hidrofobicidady altos de polaridad, flexibilidad y accesibilidad, características que le permiten a las proteínas extender su capacidad como chaperonas. La Hsp70tiene una estructura secundaria compuesta por 41-45% alfa hélices, 30-43% coil y menos del 6% en hoja plegada y la Hsp90 por 52 y 53% hélices, 26-34% coily 6% hoja plegada. Las Hsp juegan un rol importante en los insectos debido a su tamaño y corto ciclo de vida, pues la temperatura influye en su distribución y abundancia.

  5. 热休克蛋白HSP70和抑癌基因P53、PCNA在人类喉癌及癌旁组织中的表达及意义%Expression and significance of HSP70,P53 and PCNA in human,s larynx cancer

    Institute of Scientific and Technical Information of China (English)

    姚俊; 王军; 阮晓文; 罗国庆; 崔德威; 张钦明


    目的探讨热休克蛋白(heat shock Proteins,HSP)HSP70、抑癌基因突变蛋白(P53)、增殖细胞核抗原(PCNA)在人类喉癌及癌旁组织中的表达及意义.方法采用免疫组化S-P法检测HSP70蛋白、P53和PCNA在人类喉癌及癌旁组织中的表达情况,并应用统计学方法结合临床和随访资料对它们的相关性进行分析.结果HSP70、P53、PCNA的表达可能与喉癌的发生发展有关,并对喉癌的预后起指导意义.

  6. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement. (United States)

    Moghadamtousi, Soheil Zorofchian; Rouhollahi, Elham; Karimian, Hamed; Fadaeinasab, Mehran; Abdulla, Mahmood Ameen; Kadir, Habsah Abdul


    The popular fruit tree of Annona muricata L. (Annonaceae), known as soursop and graviola, is a widely distributed plant in Central and South America and tropical countries. Leaves of A. muricata have been reported to possess antioxidant and anti-inflammatory activities. In this study, the gastroprotective effects of ethyl acetate extract of A. muricata leaves (EEAM) were investigated against ethanol-induced gastric injury models in rats. The acute toxicity test of EEAM in rats, carried out in two doses of 1 g/kg and 2 g/kg, showed the safety of this plant, even at the highest dose of 2 g/kg. The antiulcer study in rats (five groups, n=6) was performed with two doses of EEAM (200 mg/kg and 400 mg/kg) and with omeprazole (20 mg/kg), as a standard antiulcer drug. Gross and histological features showed the antiulcerogenic characterizations of EEAM. There was significant suppression on the ulcer lesion index of rats pretreated with EEAM, which was comparable to the omeprazole effect in the omeprazole control group. Oral administration of EEAM to rats caused a significant increase in the level of nitric oxide and antioxidant activities, including catalase, glutathione, and superoxide dismutase associated with attenuation in gastric acidity, and compensatory effect on the loss of gastric wall mucus. In addition, pretreatment of rats with EEAM caused significant reduction in the level of malondialdehyde, as a marker for oxidative stress, associated with an increase in prostaglandin E2 activity. Immunohistochemical staining also demonstrated that EEAM induced the downregulation of Bax and upregulation of Hsp70 proteins after pretreatment. Collectively, the present results suggest that EEAM has a promising antiulcer potential, which could be attributed to its suppressive effect against oxidative damage and preservative effect toward gastric wall mucus.

  7. Responsiveness of different citrus genotypes to the Xanthomonas citri ssp. citri-derived pathogen-associated molecular pattern (PAMP) flg22 correlates with resistance to citrus canker. (United States)

    Shi, Qingchun; Febres, Vicente J; Jones, Jeffrey B; Moore, Gloria A


    The bacterial agent of citrus canker disease (Xanthomonas citri ssp. citri, Xcc) has caused tremendous economic losses to the citrus industry around the world. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is important to plant immunity. In this study, we compared the defence responses of citrus canker-resistant and citrus canker-susceptible genotypes to the Xcc-derived PAMP flg22 (Xflg22) by analysing the expression of 20 citrus defence-associated genes. We showed that, in the most resistant genotype, 'Nagami' kumquat, there was significant induction of several defence genes (EDS1, NDR1, PBS1, RAR1, SGT1, PAL1, NPR2 and NPR3) as early as 6 h and up to 72 h after Xflg22 treatment. At the other end of the spectrum, highly susceptible 'Duncan' grapefruit showed no induction of the same defence genes, even 120 h after treatment. Citrus genotypes with partial levels of resistance showed intermediate levels of transcriptional reprogramming that correlated with their resistance level. Xflg22 also triggered a rapid oxidative burst in all genotypes which was higher and accompanied by the induction of PTI marker genes (WRKY22 and GST1) only in the more resistant genotypes. Pretreatment with Xflg22 prior to Xcc inoculation inhibited bacterial growth in kumquat, but not in grapefruit. A flagellin-deficient Xcc strain (XccΔfliC) showed greater growth increase relative to wild-type Xcc in kumquat than in grapefruit. Taken together, our results indicate that Xflg22 initiates strong PTI in canker-resistant genotypes, but not in susceptible ones, and that a robust induction of PTI is an important component of citrus resistance to canker.

  8. Hsp70 inducer, 17-allylamino-demethoxygeldanamycin, provides neuroprotection via anti-inflammatory effects in a rat model of traumatic brain injury (United States)

    Gu, Youquan; Chen, Jun; Wang, Tianhong; Zhou, Chaoning; Liu, Zhaodong; Ma, Lanhua


    Traumatic brain injury (TBI) is the predominant cause of mortality in young adults and children living in China. TBI induces inflammatory responses; in addition, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 are important pro-inflammatory cytokines. Considering the observation that Hsp-70 overexpression can exert neuroprotection, identifying a drug that is able to induce the upregulation of Hsp70 has the potential to be a promising therapy for the treatment of neurological diseases. Thus, the present study assessed the clinical effectiveness of an anticancer drug and Hsp70 activator, 17-allylamino-demethoxygeldanamycin (17-AAG), to evaluate its potential as a treatment for patients with TBI. The aim of present study was to determine the neuroprotective effects of 17-AAG following trauma and to investigate the underlying mechanisms of action. To establish rat models, rats were subjected to a controlled cortical impact injury and randomly divided into vehicle or 17-AAG groups. In the 17-AAG group, rats were administered with an intraperitoneal injection of 17-AAG (80 mg/kg) immediately following the establishment of TBI. The motor function was measured using Neurologic Severity Score, and neuronal death was evaluated using immunofluorescence. The expression levels of GLT-1, Bcl-2 and Hsp-70 were detected by western blot analysis and the expression levels of inflammatory cytokines were quantified using ELISA. The present study determined that 17-AAG significantly reduced brain edema and motor neurological deficits (P<0.05), in addition to increasing neuronal survival. The aforementioned findings are associated with a downregulation of the expression levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. Conversely, no significant changes of glutamate transporter-1 expression were observed. The present results suggest that 17-AAG treatment may provide a neuroprotective effect by reducing inflammation following TBI.

  9. Nucleotides Flanking the Start Codon in hsp70 mRNAs with Very Short 5'-UTRs Greatly Affect Gene Expression in Haloarchaea.

    Directory of Open Access Journals (Sweden)

    Wenchao Chen

    Full Text Available Leaderless translation is prevalent in haloarchaea, with many of these leaderless transcripts possessing short 5'-untranslated regions (UTRs less than 10 nucleotides. Whereas, little is known about the function of this very short 5'-UTR. Our previous studies determined that just four nucleotides preceded the start codon of hsp70 mRNA in Natrinema sp. J7, with residues -3A and +4G, relative to the A of the ATG start codon, acting as the preferred bases around the start codon of all known haloarchaeal hsp70 genes. Here, we examined the effects of nucleotides flanking the start codon on gene expression. The results revealed that shortening and deletion of the short 5'-UTR enhanced transcript levels; however, it led to significant reductions in overall translational efficiency. AUG was efficiently used as start codons, in both the presence and absence of short 5'-UTRs. GUG also could initiate translation, even though it was so inefficient that it would not be detected without considerably elevated transcript. Nucleotide substitutions at position -4 to +6 were shown to affect gene expression by transcript and/or translational levels. Notably, -3A and A/U nucleotides at position +4~+6 were more optimal for gene expression. Nucleotide transversions of -3A to -3C and +4G to +4T with hsp70 promoter from either Haloferax volcanii DS70 or Halobacterium salinarum NRC-1 showed the same effects on gene expression as that of Natrinema sp. J7. Taken together, our results suggest that the nucleotides flanking the start codon in hsp70 mRNAs with very short 5'-UTRs play an important role in haloarchaeal gene expression.

  10. Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation. (United States)

    Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E


    Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

  11. Whole body cortisol and expression of HSP70, IGF-I and MSTN in early development of sea bass subjected to heat shock. (United States)

    Bertotto, Daniela; Poltronieri, Carlo; Negrato, Elena; Richard, Jacopo; Pascoli, Francesco; Simontacchi, Claudia; Radaelli, Giuseppe


    Whole body cortisol levels were determined during early larval developmental stages of sea bass (Dicentrarchus labrax) subjected to a heat shock with the aim to investigate the correlation between the stress event and the activation of the hypothalamic-pituitary-interrenal axis. Moreover, the mRNA expression of inducible heat shock protein 70 (HSP70), insulin-like growth factor I (IGF-I) and myostatin (MSTN) was also detected. Whole body cortisol was determined by a radio-immunoassay (RIA) technique whereas the expression of HSP70, IGF-I and MSTN mRNAs was quantified by Real-Time PCR. Cortisol was detectable in all the larvae from hatching but its level increased significantly in larvae submitted to heat shock from 2-day post hatching onwards. An effect of the sole transfer on cortisol levels was detectable at day 10, indicating an increase of the hypothalamic-pituitary-interrenal axis sensitivity from this stage of sea bass development. In animals exposed to heat shock, the expression of inducible HSP70 resulted in a marked increase of mRNA levels already at hatching. This increase was significantly higher from 6 days onwards if compared to controls. Moreover, heat shock resulted in a decrease (although not significant) in IGF-I mRNA expression of stressed larvae if compared to controls. On the contrary, heat shock did not influence the expression of MSTN mRNA in all groups. The results indicate a very early activation of the hypothalamic-pituitary-interrenal axis and in general of the stress response during the development of European sea bass. Moreover, these results suggest the importance of cortisol and inducible HSP70 as bioindicators of stress in aquaculture and confirm the role of IGF-I and MSTN as regulatory factors during development and growth of fish.

  12. A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in O. tsutsugamushi-Infected Human Macrophages. (United States)

    Wu, Ching-Ying; Hsu, Wen-Li; Wang, Chun-Hsiung; Liang, Jui-Lin; Tsai, Ming-Hsien; Yen, Chia-Jung; Li, Hsiu-Wen; Chiu, Siou-Jin; Chang, Chung-Hsing; Huang, Yaw-Bin; Lin, Ming-Wei; Yoshioka, Tohru


    Orientia (O.) tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF)-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE) inhibitors on TNF-α production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB), but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70) expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response.

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

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    Sri Sulistyowati


    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.

  14. Effect of ATP on the release of hsp 70 and hsp 40 from the nucleus in heat-shocked HeLa cells. (United States)

    Ohtsuka, K; Utsumi, K R; Kaneda, T; Hattori, H


    We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunofluorescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.

  15. Allele-specific PCR genotyping of the HSP70 gene polymorphism discriminating the green and red color variants sea cucumber (Apostichopus japonicus)

    Institute of Scientific and Technical Information of China (English)

    Jung-Ha Kang; Ki Hwan Yu; Jung-Youn Park; Chul-Min An; Je-Cheon Jun; Sang-Jun Lee


    Color variation is a well-known feature of sea cucumbers (Apostichopus japonicus),which are classified into three groups based on their colors of red,green and black.It is also one of the most important traits related to how they taste,and it thereby affects their market price.Attempts were made to identify single-nucleotide polymorphisms (SNPs) and to analyze differences associated with SNP genotypes between green and red color variants using HSP70 as the target gene.The HSP70 gene,which is found universally in organisms from bacteria to humans,is one of the most evolutionarily conserved genes and the most widely studied biomarker of stress response.DNA fragments of 1074 bp covering a partial sequence of the sea cucumber HSP70 gene,were amplified from both red and green variants,and subsequently analyzed for the presence of SNPs.Twenty-seven polymorphic sites in total,including heterozygous sites,were observed.Of these,six sites were found to be significantly different SNP genotypes between green and red variants.Furthermore,PCR with an internal primer designed to include an allelespecific SNP at the 3' end (site 443) showed differentiation between the two variants,100% and 4.2% amplification in green and red variants,respectively.The validated SNPs may serve as informative genetic markers that can be used to distinguish variants at the early developmental stage,prior to color differentiation.

  16. 基于hsp70mRNA的RT-qPCR方法评估水中病源微生物的灭活效果%Assessing the inactivation of pathogenic microorganisms in water based on reverse transcription quantitative PCR with hsp70 mRNA

    Institute of Scientific and Technical Information of China (English)

    侯阳阳; 冉治霖; 李绍峰



  17. 温室粉虱和烟粉虱3个隐种中热激蛋白基因hsp70和hsp90含量的比较分析%Comparative analysis of the copy numbers of hsp70 and hsp90 in Trialeurodes vaporariorum and three cryptic species of Bemisia tabaci complex (Hemiptera: Aleyrodidae)

    Institute of Scientific and Technical Information of China (English)

    韦姣; 吕志创; 王韧; 万方浩


    [目的]昆虫适应新环境的能力与其对温度的耐受能力密切相关.热激蛋白HSP70和HSP90具有提高生物体温度耐受性的功能.烟粉虱Bemisia tabaci (Gennadius)不同隐种和不同种粉虱对温度的适应能力有差异,这与它们的热激蛋白基因拷贝数的差异可能相关.[方法]利用实时荧光定量PCR方法,检测入侵型烟粉虱MED隐种和MEAM1隐种、本地型烟粉虱AsiaⅡ1隐种以及温室粉虱Trialeurodes vaporariorum (Westwood)基因组DNA中热激蛋白基因hsp70和hsp90的拷贝数.[结果]不同种类的粉虱和烟粉虱不同隐种体内的hsp70和hsp90的含量均有较大差异,其中hsp70和hsp90拷贝数在入侵型烟粉虱MED和MEAM1隐种中含量较其他两种均高,而在土著种AsiaⅡ1隐种中含量最低,在温室粉虱中居中.此外,相同物种雌雄成虫hsp70和hsp90的拷贝数也不同,雌虫hsp70和hsp90拷贝数约为雄虫的2倍.[结论]不同种粉虱及烟粉虱不同隐种的hsp70和hsp90的拷贝数可能与其耐热性差异相关.本研究为解释不同种粉虱、烟粉虱不同隐种及其不同性别的耐热性差异机制提供了进一步的依据.

  18. COPD and levels of Hsp70 (HSPA1A) and Hsp27 (HSPB1) in plasma and lymphocytes among coal workers: a case-control study. (United States)

    Cui, Xiuqing; Xing, Jingcai; Liu, Yuewei; Zhou, Yun; Luo, Xin; Zhang, Zhihong; Han, Wenhui; Wu, Tangchun; Chen, Weihong


    This case-control study aimed to investigate whether the levels of Hsp70 (HSPA1A) and Hsp27 (HSPB1) in plasma and lymphocytes were associated with the risk of chronic obstructive pulmonary disease (COPD) among coal workers. A total of 76 COPD cases and 48 age-matched healthy controls from a group of coal workers were included. The case group consisted of 35 COPD patients whose condition was complicated with coal workers' pneumoconiosis (CWP) and 41 COPD patients without CWP. Heat shock proteins (Hsps) in plasma and lymphocytes were detected by ELISA and flow cytometry, respectively. Multiple logistic regression models were applied to estimate the association between Hsp levels and COPD risk. Our results showed that plasma Hsp70 and lymphocyte Hsp27 levels were significantly higher and plasma Hsp27 levels were significantly lower in COPD cases than in controls (p < 0.01). No significant differences in lymphocyte Hsp70 levels were found between COPD cases and the matched subjects. Higher plasma Hsp70 levels (odds ratio (OR) = 13.8, 95 % confidence interval (CI) = 5.7-33.5) and lower plasma Hsp27 levels (OR = 4.6, 95 % CI = 2.0-10.5) were significantly associated with an increased risk of COPD after adjusting for confounders. Higher lymphocyte Hsp27 levels were only associated with an increased risk of COPD with CWP (OR = 6.6, 95 % CI = 2.0-22.1) but not with an increased risk of COPD without CWP (OR = 3.0, 95 % CI = 0.9-8.9). Additionally, there were strong joint effects of different Hsps on COPD risk. These results showed that higher levels of plasma Hsp70 and lower levels of plasma Hsp27 might be associated with an increased risk of COPD among coal workers. They may have the potential to serve as monitoring markers for COPD in coal workers.

  19. Expression of hsp70, hsp100 and ubiquitin in Aloe barbadensis Miller under direct heat stress and under temperature acclimation conditions. (United States)

    Huerta, Claudia; Freire, Matías; Cardemil, Liliana


    KEY MESSAGE : The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35 °C prior to a heat shock of 45 °C. Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40 °C and annual precipitation of 40 mm (dry years) to 170 mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT(50) of Aloe vera was determined as 53.2 °C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95 %, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40-45 °C increased 50 % over the controls, while the expression increased by 150 % over the controls under a water deficit of 50 % FC. The expression of all three genes was also studied under 2 h of acclimation at 35 or 40 °C prior to a heat shock at 45 °C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.

  20. Construction of Eukaryotic Coexpression Vector Containing Mycobacterium Tuberculosis Heat Shock Protein 70 and Herpes Simplex Virus Thymidine Kinase Genes%mtHSP70/HSV-TK双基因真核共表达质粒的构建

    Institute of Scientific and Technical Information of China (English)

    曾曙光; 刘启才; 王素文; 徐平平; 章锦才; 张积仁


    目的 构建结核杆菌热休克蛋白70(mycobacterium tuberculosis heat-shock proteins 70,mtHSP70)和自杀基因单纯疱疹病毒-胸苷激酶(herpes simplex virus-thymidinekinase,HSV-TK)双基因真核共表达质粒pCMV-mtHSP70-IRES-TK.方法 登录Genbank查询HSV-TK和mtHSP70基因mRNA序列,应用引物设计软件DNA club分别设计扩增HSV-TK和mtHSP70基因全长cDNA的特异引物.以pcDNA3-TK质粒或mtHSP70质粒DNA为模板,分别采用各自的引物,用prime STAR HS DNA Polymerase进行聚合酶链反应(polymerase chain reaction,PCR),获得HSV-TK和mtHSP70基因cDNA片断,连接到T载体pMD18-T上,转化大肠杆菌TG1后用HSV-TK和mtHSP70基因的特异引物进行菌落PCR,确定含有阳性mtHSP70或HSV-TK重组质粒并将阳性pMD18T-TK重组质粒和pMD18T-mtHSP70重组质粒进行上、下游测序,测序结果与基因序列进行同源比较.选定正确的质粒用于HSV-TK和mtHSP70基因的亚克隆,构建质粒PCMV-mtHSP70-IRES-TK,所得阳性克隆摇菌后少量提取质粒,分别用NotⅠ单酶切及EcoRⅠ和XhoⅠ双酶切进行酶切鉴定.结果 构建的pCMV-mtHSP70-IRES-TK质粒分别用NotⅠ单酶切及EcoRⅠ/XhoⅠ双酶切进行酶切鉴定,酶切产物电泳见特异酶切图谱,确定为重组质粒pCMV-mtHSP70-IRES-TK.结论 本实验为后续研究mtHSP70/HSV-TK双基因的协同抗肿瘤作用奠定了实验基础.%Objective To construct an eukaryotic plasmid PCMV-mtHSP70-IRES-TK that contains and expresses both mycobacterium tuberculosis heat shock protein 70 (mtHSP70) gene and suicide gene HSV-TK for the experiments. Methods Login on genbank and search for mRNA sequences of HSV-TK and mtHSP70 genes. Specific primers for the amplification of full length cDNA of HSV-TK and mtHSP70 were designed by primer designing software DNA club. pcDNA3-TK plasmid and mtHSP70 plasmid DNA were used as template. PCR was performed with respective primers and prime STAR HS DNA polymerase, cDNA segments of HSV-TK and mtHSP70

  1. Specific anti-tumor immune responses of dendritic cells pulsed with recombinant human rhHSP70 and freeze-thaw cellular lysates derived from breast cancer%rhHSP70联合冻融抗原修饰树突状细胞诱导的抗乳腺癌作用*

    Institute of Scientific and Technical Information of China (English)

    李斌; 陈鹏; 郑建云


      目的:利用rhHSP70联合树突状细胞递呈肿瘤抗原的特性提高细胞毒T淋巴细胞(CTLs)对乳腺癌细胞的杀伤活性。方法:外周血单个核细胞体外经GM-CSF和IL-4诱导产生树突状细胞,负载冻融抗原肽的同时加入新型热休克蛋白(rhHSP70),不同分组分别诱导自体CTLs产生。ELISA测定CTLs杀伤活性和细胞因子的分泌。结果:冻融抗原肽致敏的DCs促进CTLs增殖,上调CTLs中CD3+和CD8+T细胞群及Th1型细胞因子的分泌;体外实验中具有对人乳腺癌细胞MCF-7的杀伤活性,在加入rhHSP70后效果更加明显,并能显著增强CTLs对肿瘤细胞的杀伤率。结论:rhHSP70联合肝癌冻融抗原修饰DCs,能够促进DCs的成熟,增强DCs刺激淋巴细胞增殖的能力,诱导的CTLs在体外对乳腺癌细胞能产生高效杀伤力。rhHSP70增强DCs抗肿瘤能力的机制可能与其促进DCs成熟有关。%Objective:This work aims to use the characteristics of dendritic cells (DCs) pulsed with recombinant human HSP70, which can present and process tumor antigens, to enhance the killing activity of cytotoxic t lymphocytes (CTLs) against breast neoplasms. Methods:Autologous DCs were isolated from peripheral blood mononuclear cells and then stimulated in vitro with granulocyte macrophage-colony stimulating factor and interleukin-4. The DCs were loaded with A549 tumor cell freeze-thaw lysate, and rhHSP70 was added as an immune adjuvant. The specific groups were subjected to tumor-specific cytotoxic assay, enzyme-linked immunosorbent assay, and fluores-cence-activated cell sorting. Results:DCs pulsed with A549 tumor cell lysate enhanced the growth expansion of CTLs, upregulated CD40 and CD80 populations in CTLs, and augmented Th1 cytokines. In addition, the cytotoxicity of specific CTLs against A549 was highly enhanced. The above indications became more obvious after the addition of rhHSP70. Conclusion:DCs pulsed with freeze-thaw cell

  2. 中耳细菌感染诱导的急性期HSP-70相关反应的免疫组织化学研究%Immunocytochemical research of HSP-70 correlative response in acute stage induced by bacterial infected middle ear

    Institute of Scientific and Technical Information of China (English)

    周永清; 邹静; 毕爱芳; 尚耀东


    探讨中耳细菌感染急性期时,哺乳动物中、内耳热休克反应的部位,以及中耳细菌感染诱生的热休克蛋白是否可能引发内耳自身免疫损伤。运用中耳注射肺炎克雷伯杆菌制成豚鼠中耳急性感染动物模型,分别于接种后第1、3、5、7天处死动物取材。应用免疫组化技术,研究了中耳粘膜和耳蜗表达热休克蛋白70(HSP-70)的部位。结果表明:正常状态下,中耳粘膜表层的上皮细胞和内耳膜迷路血管纹、螺旋韧带、Corti氏器均有弱的阳性反应,感染应激后,上述同样部位均有强的阳性显色。不同取材时段显示的阳性位置无差异。说明在中耳急性细菌感染期,中耳粘膜和内耳组织均表达了同源HSP-70蛋白分子,这些同源HSP-70为引发内耳自身免疫损伤提供了物质基础。%To illustrate the location of HSP-70 (heat\\|shock protein\\|70)correlative response in acute stage between middle and inner ear when mammalian middle ear infected by bacteria, and investigate the possibility to cause autoimmune inner ear injure by HSP-70 that induced by otitis media. An animal model of acute otitis media was established by inoculating Klebsiella pneumoniae (K.p) into middle ear of guinea pigs. Animals were sacrificed 1,3,5 and 7days after inoculation.Investigation of the locations of HSP-70 in middle ear mucosa and cochlea structures were detected by immunocytochemical technique .The results showed that light stain of HSP-70 existed in the superficial epithelium cells of normal mucosa and cochlea structures such as stria vascularis, spiral ligament and Corti′s organ.Heavy stain appeared in the same sites in the infected ears . No different positive sites was observed in all animals.Our findings suggested that HSP-70 were expressed in middle ear mucosa and inner ear structures during acute middle ear infective stage.The HSP-70 provide a material base for causing autoimmune inner ear injure

  3. Transcriptional responses of xenobiotic metabolizing enzymes, HSP70 and Na+/K+ -ATPase in the liver of rabbitfish (Siganus oramin) intracoelomically injected with amnesic shellfish poisoning toxin. (United States)

    Wang, Lin; Liang, Xu-Fang; Huang, Yan; Li, Shi-Ying; Ip, Kok-Chao


    Amnesic shellfish poisoning toxin domoic acid (DA) is a marine neurotoxin that accumulates in fish and shellfish, and has been implicated to be involved in human and marine wildlife mortality. The transcriptional responses of cytochrome P-450 1A (CYP1A), glutathione S-transferase alpha (GSTA), glutathione S-transferase rho (GSTR), heat shock protein 70 (HSP70), and Na(+)/K(+)-ATPase alpha 1 (ATP1A1) in the liver of rabbitfish (Siganus oramin) intracoelomically injected with DA, were investigated. Experimental fish were administered with one injection of DA (2 microg/g wet weight) or PBS as control. After 24 h, fish were killed and hepatic RNA was isolated. Partial cDNA of rabbitfish CYP1A, GSTA, GSTR, HSP70, ATP1A1, and beta-actin were obtained by PCR using degenerate primers. Using beta-actin as an external control, the relative liver CYP1A, GSTA, GSTR, HSP70, and ATP1A1 mRNA abundance of rabbitfish were determined by semi-quantitative RT-PCR within the exponential phase. The ratio CYP1A/beta-actin mRNA (%) of exposure group was determined to be 148.92+/-12.69, whereas the ratio of control group was 82.3+/-8.35, indicating that CYP1A was induced significantly in rabbitfish following DA exposure (P0.05). The induction of hepatic CYP1A in response to DA suggests a potential role for fish phase I xenobiotic metabolizing enzyme in DA metabolism.

  4. Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90,GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

    Institute of Scientific and Technical Information of China (English)

    Seung Oe Lim; Cheol Keun Park; Sung Gyoo Park; Jun-Hi Yoo; Young Min Park; Hie-Joon Kim; Kee-Taek Jang; Jae Won Cho; Byung Chul Yoo; Gu-Hung Jung


    AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)related HCC.METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucoseregulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed.RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis.CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBVrelated hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

  5. Divergent ecological histories of two sister Antarctic krill species led to contrasted patterns of genetic diversity in their heat-shock protein (hsp70) arsenal. (United States)

    Papot, Claire; Cascella, Kévin; Toullec, Jean-Yves; Jollivet, Didier


    The Arctic and the Antarctic Peninsula are currently experiencing some of the most rapid rates of ocean warming on the planet. This raises the question of how the initial adaptation to extreme cold temperatures was put in place and whether or not directional selection has led to the loss of genetic variation at key adaptive systems, and thus polar species' (re)adaptability to higher temperatures. In the Southern Ocean, krill represents the most abundant fauna and is a critical member at the base of the Antarctic food web. To better understand the role of selection in shaping current patterns of polymorphisms, we examined genetic diversity of the cox-1 and hsp70 genes by comparing two closely related species of Euphausiid that differ in ecology. Results on mtcox-1 agreed with previous studies, indicating high and similar effective population sizes. However, a coalescent-based approach on hsp70 genes highlighted the role of positive selection and past demographic changes in their recent evolution. Firstly, some form of balancing selection was acting on the inducible isoform C, which reflected the maintenance of an ancestral adaptive polymorphism in both species. Secondly, E. crystallorophias seems to have lost most of its hsp70 diversity because of a population crash and/or directional selection to cold. Nonsynonymous diversities were always greater in E. superba, suggesting that it might have evolved under more heterogeneous conditions. This can be linked to species' ecology with E. superba living in more variable pelagic conditions, while E. crystallorophias is strictly associated with continental shelves and sea ice.

  6. Early infection dynamics after experimental challenge with Mycobacterium avium subspecies paratuberculosis in calves reveal limited calf-to-calf transmission and no impact of Hsp70 vaccination. (United States)

    Santema, Wiebren J; Poot, Jacqueline; Segers, Ruud P A M; Van den Hoff, Daniëlle J P; Rutten, Victor P M G; Koets, Ad P


    Efficient control of bovine paratuberculosis is hampered by lack of a vaccine. The purpose of this study was to evaluate efficacy of a candidate vaccine, consisting of recombinant Mycobacterium avium subspecies paratuberculosis (MAP) Hsp70 with DDA adjuvant, in calves experimentally infected with MAP. Four groups of 14 animals each were used. Animals in group 1 and 2 were all vaccinated with Hsp70/DDA at day 0, 84, 168 and 357, and those in group 3 and 4 were non-vaccinated controls. In each group half (n=7) of the animals were challenged and the remaining half served as contacts. Blood and fecal samples were collected at three week intervals until day 588, and subsequently all animals were subjected to necropsy. The primary outcomes assessed were fecal culture (FC) of MAP, tissue colonization of MAP, and transmission of infection to contact animals. The kinetics of MAP shedding in feces of challenged animals showed a peak around 130 days post-challenge, irrespective of vaccination status. At necropsy no differences in the level of tissue colonization between vaccinated animals and controls were observed in the challenged groups. Only one contact animal (non-vaccinated) was positive at necropsy, indicating limited to no transmission within groups. These findings indicate that Hsp70/DDA vaccination does not influence early infection dynamics after experimental infection. However, early shedding of MAP in calves did not result in efficient transmission of infection to contact animals. The latter implies that introduction of an infected calf in a cohort of susceptibles has limited consequences for spread of infection.

  7. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It's Antiserum%细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    赵莉; 薛晶; 石保新; 陈皓斐; 张文宝; 马正海; 张壮志; 张旭; 古努尔·吐尔逊; 米晓云; 金映红


    [Objective] The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. [ Methods] EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum's specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. [Results] Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol· L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1 : 256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. [Conclusion] The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.%[目的]克隆细粒棘球绦虫(Echinococcus granulosus,E.g)热休克蛋白家族基因Hsp70,在原核细胞中表达、纯化Hsp70蛋白并制

  8. Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function. (United States)

    Wadhwa, R; Sugihara, T; Yoshida, A; Nomura, H; Reddel, R R; Simpson, R; Maruta, H; Kaul, S C


    MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.

  9. Oxygen glucose deprivation post-conditioning protects cortical neurons against oxygen-glucose deprivation injury: role of HSP70 and inhibition of apoptosis. (United States)

    Zhao, Jian-hua; Meng, Xian-li; Zhang, Jian; Li, Yong-li; Li, Yue-juan; Fan, Zhe-ming


    In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.

  10. A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter*



    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase...

  11. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis. (United States)

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha


    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody


    Institute of Scientific and Technical Information of China (English)

    孙勇; 章力; 李慕婵; 吴任; 雷腊梅; 谢数涛


    Red swamp crayfish, Procambarus clarkia, with advantages of easy cultivation and availability, can live in various tough environments, so intrigued researchers pay attention to its ecology, toxicology, physiology and immunology. We studied an inducible cDNA encoding HSP70 in P. clarkia which would contribute to extensive researches of HSP70s and environmental stresses. An inducible heat shock protein 70 (HSP70) cDNA was cloned from red swamp crayfish by RTPCR and RACE, which named scHSP70. The full-length cDNA of the scHSP70 was 2271 bp, consisting of a partial 5'-terminal untranslated region (UTR) of 142bp, a 3'-terminal UTR of221bp, an open reading frame of 1902bp (ORF) and a poly (A) tail and GenBank No. DQ301506. The gene contained only one exon according to amplification of scHSPT0 from genomic DNA. The scHSP70 cDNA encoded a polypeptide of 635 amino acids. Based on phylogenetic analysis, the gene was clustered with inducible HSP70 family members from other species. The evolution relationship was consisted with traditional classification. Semi-quantitative PCR was employed to assess the temporal expression of scHSP70 mRNA levels from heat-shock treated and unstressed crayfish. Challenge of the red swamp crayfish with 2h heat shock resulted in dramatic increases in the expression of HSP70 mRNA levels in all tissues, heart, muscle, hemocytes, digestive gland, antenhal gland, testis and intestine, among which, the highest expression was found in heart. However, under normal conditions, the expressions of HSP70 mRNA level were much lower in all tissues compared to treated ones, especially in haemocytes. The upregulated mRNA expression of the HSP70 in the crayfish following heat shock indicates that scHSP70 gene is inducible. These stress proteins provide invaluable information in stress response in the crayfish.%克氏原螯虾是我国淡水虾类养殖的重要品种,具有很强的抵御各种环境胁迫和各种刺激的能力.本文以该虾为对象,通过

  13. Evaluation of the toxic potential of calcium carbide in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). (United States)

    Danish, Mohd; Fatima, Ambreen; Khanam, Saba; Jyoti, Smita; Rahul; Ali, Fahad; Naz, Falaq; Siddique, Yasir Hasan


    In the present study the toxic potential of calcium carbide (CaC2) was studied on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). The third instar larvae were exposed to 2, 4, 8, 16 and 32×10(-3)g/ml of CaC2 in diet for 24h. The results reveal that the dose 2×10(-3)g/ml was not toxic but the remaining doses showed a dose dependent significant increase in the hsp70 expression, β-galactosidase activity, tissue damage, oxidative stress markers (lipid peroxidation and protein carbonyl content), glutathione-S-transferase activity, expression of Caspase 3 and 9, apoptotic index and DNA damage (midgut cells). A significant reduction as compared to control group in total protein, glutathione content and acetylcholinesterase activity was also observed. The Inductively Coupled Plasma Atomic Emission Spectroscopy analysis (ICPAES) reveals the presence of copper, iron, sodium, aluminium, manganese, calcium, nickel and mercury. The toxic effects of CaC2 in the present study may be attributed to the impurities present in it.

  14. The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following different administration routes. (United States)

    Ge, Wei; Hu, Pei-Zhen; Huang, Yang; Wang, Xiao-Ming; Zhang, Xiu-Min; Sun, Yu-Jing; Li, Zeng-Shan; Si, Shao-Yan; Sui, Yan-Fang


    Our previous study showed that nanoemulsion-encapsulated MAGE1-HSP70/SEA (MHS) complex protein vaccine elicited MAGE-1 specific immune response and antitumor effects against MAGE-1-expressing tumor and nanoemulsion is a useful vehicle with possible important implications for cancer biotherapy. The purpose of this study was to compare the immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70 and SEA as NE(MHS) vaccine following different administration routes and to find out the new and effective immune routes. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. C57BL/6 mice were immunized with NE(MHS) via po., i.v., s.c. or i.p., besides mice s.c. injected with PBS or NE(-) as control. The cellular immunocompetence was detected by ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were also examined. The results showed that the immune responses against MAGE-1 expressing murine tumors elicited by NE(MHS) via 4 different routes were approximately similar and were all stronger than that elicited by PBS or NE(-), suggesting that this novel nanoemulsion carrier can exert potent antitumor immunity against antigens encapsulated in it. Especially, the present results indicated that nanoemulsion vaccine adapted to administration via different routes including peroral, and may have broader applications in the future.

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

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


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

  16. [Role of heat-shock proteins of Hsp70 family in changes of narcotic activity of hyperbaric nitrogen during increasing hypoxic stimulus]. (United States)

    Vetosh, A N; Kostkin, V B; Alekseeva, O A; Korzhevskiĭ, D E


    The spontaneous motor activity and pose reflexes of male adult rats (Wistar) were observed in the course of high pressure nitrogen compression up to 4,1 MPa. The experiments were carried out under normoxic and hypoxic conditions. Stabile rat motor cortex oxygen tension was recording during the nitrogen compression up to 7,1 MPa under normoxic condition. Sensitivity to nitrogen high pressure to be on the increase under hypoxic conditions. In its turn, resistibility to nitrogen high pressure to be on the decrease under hypoxic conditions (oxygen partial pressure from 0,012 to 0,004 MPa). Quantity of high dencity heat shock proteins (Hsp70) rats motor cortex neurons was 3,44 times higher after course of high pressure nitrogen compression up to 4,1 MPa. For hypoxic exposure (6% O2) the difference was less pronounced - 2,2 times. Data about rat motor cortex neurons Hsp70 concentration under high nitrogen pressure and low oxygen pressure may turn to be a clear base for explanation hypoxic influence on processes of nitrogen narcosis.

  17. Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1. (United States)

    Han, Sumin; Liu, Yu; Chang, Amy


    Cells have a variety of strategies for dealing with misfolded proteins. Heat shock response involves transcriptional induction of chaperones to promote and/or correct folding, and also activation of the ubiquitin/proteasome system to degrade defective proteins. In the secretory pathway, it is primarily luminal misfolded or unassembled proteins that trigger the unfolded protein response which, like heat shock, induces chaperones and components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. To understand cellular response to a misfolded polytopic membrane protein of the secretory pathway, we studied Pma1-D378S, a model ERAD substrate. Expression of misfolded Pma1 induces heat shock response in the absence of increased temperature. Overexpression of HSF1, the transcription factor that mediates heat shock response, increases degradation of Pma1-D378S without temperature upshift. Nevertheless, efficient Pma1-D378S degradation occurs in an hsf1 mutant that maintains basal transcription levels but cannot mediate transcriptional activation. Thus, heat shock protein induction enhances but is not necessary for ERAD. The Ssa group of cytoplasmic Hsp70 chaperones is required for ERAD of both Pma1-D378S and another transmembrane ERAD substrate, Ste6*. In the absence of Ssa chaperones, ubiquitination of both substrates is impaired, resulting in stabilization. We suggest a role for Hsp70 cytoplasmic chaperones in recognition by the endoplasmic reticulum-associated ubiquitination machinery.

  18. HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis (United States)

    Gruppi, C. M.; Wolgemuth, D. J.


    This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

  19. Cytotoxicity and Expression of c-fos, HSP70, and GADD45/153 Proteins in Human Liver Carcinoma (HepG2 Cells Exposed to Dinitrotoluenes

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou


    ŽÂ¼g/mL or within the dose range of 0-200 μg/mL for 2,6-DNT. The 45-kDa growth arrest and damage protein was significantly expressed at the dose range of 200 – 250μg/mL for 2,6-DNT and at the dose range of 200 - 400μg/mL for 2,4-DNT. Expression of 153-kDa growth arrest and DNA damage protein was significant at the 100, 200, and 250μg/mL doses for 2,6-DNT and at the 200 μg/mL dose for 2,4-DNT. Overall, these results indicate the potential of DNTs to induce cytotoxic, proteotoxic (HSP70, and genotoxic (GADD45/153 effects, as well as oxidative stress and pro-inflammatory reactions (c-fos.

  20. Changes of Thermoregulatory Responses and HSP70 in 10-day Heat Acclimation of Middle-Long-Distance Runners%中长跑运动员10天热适应过程中机体热调节反应及HSP70变化

    Institute of Scientific and Technical Information of China (English)

    吴卫兵; 王人卫; 许弟群


    目的:观察10天热适应过程中机体热调节反应及HSP70变化,探讨运动员运动训练和比赛实践中热适应的有效建立.方法:以8名中长跑男性运动员为受试对象,在温度为33℃、湿度为80%RH环境条件下,进行连续10天60 min、40%VO2 max强度的热适应运动,观察第2天、6天和10天机体热调节反应及HSP70变化.结果:中长跑运动员10天热适应过程中的体核温度、心率、主观感觉疲劳程度、出汗量、出汗率、汗液电解质离子和HSP70出现一些明显的变化,主要表现为热适应过程中运动后体核温度、心率逐渐下降,运动排汗能力增强,汗液电解质离子浓度显著下降(P<0.01),运动后HSP70水平显著升高(P<0.01).结论:热适应过程中机体出现体核温度下降、心率下降、排汗能力增强和HSP70水平升高等热适应性变化,提示中长跑运动员经过10天热适应运动后,机体已经有效地建立了热适应.

  1. In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

    Energy Technology Data Exchange (ETDEWEB)

    Ebrahimi, Seyyed Mahmoud, E-mail: [Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O. Box 14155-3651,Tehran (Iran, Islamic Republic of); Research Center of Virus and Vaccine, Baqiyatallah University of Medical Science, P.O.Box 14155-3651, Tehran (Iran, Islamic Republic of); Dabaghian, Mehran [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of); Tebianian, Majid [Department of Biotechnology, Razi Vaccine and Serum Research Institute (RVSRI), P.O. Box 31975/148, Karaj, Tehran (Iran, Islamic Republic of); Zabeh Jazi, Mohammad Hossein [Department of Pathobiology, University of Tehran, Faculty of Veterinary Medicine, P.O. Box 14155-6453, Tehran (Iran, Islamic Republic of)


    Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS-PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0-33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

  2. Review article: Heat shock protein 70 (Hsp70 peptide activated Natural Killer (NK cells for the treatment of patients with non-small cell lung cancer (NSCLC after radiochemotherapy (RCTx – from preclinical studies to a clinical phase II trial

    Directory of Open Access Journals (Sweden)

    Hanno M Specht


    Full Text Available Heat shock protein 70 (Hsp70 is frequently overexpressed in tumor cells. An unusual cell surface localization could be demonstrated on a large variety of solid tumors including lung, colorectal, breast, squamous cell carcinomas of the head and neck, prostate and pancreatic carcinomas, glioblastomas, sarcomas and hematological malignancies, but not on corresponding normal tissues. A membrane (mHsp70-positive phenotype can be determined either directly on single cell suspensions of tumor biopsies by flow cytometry using cmHsp70.1 monoclonal antibody or indirectly in the serum of patients using a novel lipHsp70 ELISA. A mHsp70-positive tumor phenotype has been associated with highly aggressive tumors, causing invasion and metastases and resistance to cell death. However, natural killer (NK, but not T cells were found to kill mHsp70-positive tumor cells after activation with a naturally occurring Hsp70 peptide (TKD plus low dose IL-2 (TKD/IL-2. Safety and tolerability of ex vivo TKD/IL-2 stimulated, autologous NK cells has been demonstrated in patients with metastasized colorectal and NSCLC in a phase I clinical trial. Based on promising clinical results of the previous study, a phase II randomized clinical study was initiated in 2015. The primary objective of this multicenter proof-of-concept trial is to examine whether an adjuvant treatment of NSCLC patients after platinum based radiochemotherapy with TKD/IL-2 activated, autologous NK cells is clinically effective. As a mHsp70-positive tumor phenotype is associated with poor clinical outcome only mHsp70-positive tumor patients will be recruited into the trial. The primary endpoint of this study will be the comparison of the progression-free survival of patients treated with ex vivo activated NK cells compared to patients who were treated with radiochemotherapy alone. As secondary endpoints overall survival, toxicity, quality-of-life and biological responses will be determined in both study groups.

  3. 苯并[a]芘对原代培养大鼠神经元细胞损伤及Hsp70表达的影响%The influence of Berzo [a]pyrene on the damage of primary cultured rat nerve cells and Hsp70 expression

    Institute of Scientific and Technical Information of China (English)

    许国耀; 郑金平; 王春芳; 李鹏飞; 蔚洪恩


    Objective To study the effect of benzo [ a ] pyrene (B[a]P) on damage and Heat shock protein 70(Hsp70) expression in primary culture nerve cells from rats. Methods Cortical nerve cells of SD rats (aged within 3 days) were isolated and cultured, which were exposed by different doses of B[a]P and S9. The morphological changes of nerve cells were observed by invert microscope. MTT assay was used to detect cell viability. The LDH activity and MDA concentration were detected by ultraviolet spectrophotometer. The cell apoptosis rate was detected by flow cytometry. DNA damage was detected by single cell gel electrophoresis. The expression of Hsp70 was measured by Western blot. Results After 24 h exposed by B[a]P + S9, the numbers of axons and dendrites of nerve cells were decreased, the length of synapses was shortened, some cells became vacuolated and the cell viability decreased. The LDH activity,MDA concentration in cell culture medium, Olive tail and apoptosis rate of nerve cell increased with doses of B[ a] P, and there were significant differences among 0. 5 ~10 μmol/L groups and control group. The expression of Hsp70 in 0. 5 ~ 10 μmol/L groups decreased and the difference was significant ( P < 0.05) compared with 0. 1 μmol/L group, and it was lower in 10 μmol/L group than control group (P <0.05). Simple correlation analysis indicated that there had a positive correlation between MDA,LDH, Olive tail, and cell apoptosis rate, while a negative correlation between MDA,Hsp70, and cell viability (A value). The Hsp70 had a negative correlation with LDH, Olive tail and cell apoptosis rate, while a positive correlation with cell viability (A value). A similar positive correlation existed between LDH, Olive tail and cell apoptosis rate. Conclusion B[ a] P metabolites could lead to the injury and apoptosis of primary culture of nerve cells from rats and inhibited the expression of Hsp70, which might relate to B[a]P neurotoxicity.%目的 研究苯并[a]芘(B[a]P)对体外

  4. Effects of propofol anesthesia on serum interleukins and HSP70 levels in patients with liver cancer after radiofrequency ablation%丙泊酚麻醉对肝癌射频消融术患者血清白介素和HSP70水平的影响

    Institute of Scientific and Technical Information of China (English)

    冯惠民; 李廷坤; 吕帅国; 冯艳平; 李长生


    目的:探讨丙泊酚全身麻醉对肝癌射频消融术患者血清白介素(IL)-6、8、10和热休克蛋白70(HSP70)水平的影响.方法:择期行肝癌射频消融术的患者40例,分成2组施术,每组20例.观察组患者在全麻诱导后应用丙泊酚和瑞芬太尼维持麻醉,对照组患者在穿刺部位利多卡因局部阻滞麻醉.术中检测麻醉前、首次穿刺成功时,热封闭针道时,术后1 h、24 h和48 h时的平均血压、心率、脉搏血氧饱和度和体温;采集患者术前,术后1、24和48 h静脉血5 mL,ELISA法测定血清IL-6、IL-10、IL-8及HSP70.结果:2组患者术中和术后体温、血压均升高,但观察组变化幅度小于对照组(体温:F组间=14.568,F时间=291.525,F交互=13.655,P均<0.001;血压:F组间=23.372,F时间=14.007,F交互=25.795,P均<0.001).2组术后血清IL-6、8、10及HSP70水平亦较术前升高(F时间分别为48.793、53.721、25.970和24.779,P均<0.001),而观察组L-6、8和HSP70升高幅度小于对照组(F组间=31.953、59.836和20.479,P均<0.001).结论:丙泊酚麻醉可减轻肝癌射频消融术患者的伤害性炎性反应.%Aim;To study the effects of propofol anesthesia on serum interleukins (IL) and HSP70 levels in patients with liver cancer after radiofrequency ablation ( RFA). Methods:Forty ASA I-Ⅱ patients with liver cancer aged 40 ~65 years would receive RFA were divided into 2 groups (20 in each group). Patients in group P were given propofol to maintain general anesthesia. Patients in group C were given lidocaine to maintain local anesthesia . MBP, HR, SPO2, and temperature were detected at the time of pre -anesthesia , post-anesthesia, heat blocking action , and 1,24 and 48 h after therapy. In addition , serum IL-6, IL-8, IL-10 and HSP70 levels were determined before therapy and at 1,24, and 48 h after therapy by ELISA. Results: The temperature , MBP, and serum HSP70, IL-6, IL-8 , IL-10 levels of the two groups increased during RFA (Ftime =291

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

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail:


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

  6. HSP70和NF-κB在大鼠肾缺血再灌注损伤中的表达及洛伐他汀的干预研究%Intervention of Lovastatin in the expression of HSP70 and NF-κB after renal ischemia-reperfusion injury in rats

    Institute of Scientific and Technical Information of China (English)

    李鹏; 郝雷; 高进; 傅亮


    目的 探讨洛伐他汀(Lovastatin,Lov)对大鼠肾缺血再灌注损伤(RIRI)热休克蛋白70 (HSP70)和核因子-κB (NF-κB)表达的影响.方法 90只Wistar大鼠随机分为3组:假手术(Sham)组、缺血再灌注模型(IR)组、洛伐他汀(Lov)组.IR组和Lov组大鼠采用夹闭肾动脉60 min后再灌注的方法制备RIRI模型,Lov组于动脉夹闭前给予洛伐他汀2mg/(kg·d)灌胃,各组大鼠在再灌注4、12、24 h时分别处死10只取标本.测定各组大鼠的血肌酐(Scr)、尿素氮(BUN)水平,免疫组化法检测肾组织HSP70和NF-κB表达,镜下观察肾组织形态学改变.结果 与Sham组大鼠相比,IR组大鼠在再灌注4、12、24 h时点的Scr和BUN含量均显著升高;肾组织HSP70和NF-κB表达均明显增强,差异均有统计学意义(P均<0.05).与IR组相比,Lov组大鼠在各时点的Scr和BUN含量均显著降低(P均<0.01);HSP70表达均升高(P均<0.05);NF-κB表达减少,再灌注4、12 h时,两者差异有统计学意义(P<0.05);镜下病理改变减轻.结论 Lov可通过增加肾脏HSP70、下调NF-κB达而抑制炎症反应过程,减轻肾脏损害,对大鼠RIRI起到一定的保护作用.%Objective To investigate the possible effects of Lovastatin on the expression of HSP70 and NF-KB in rats after renal ischemia-reperfusion injury (RIRI). Methods Ninety Wistar rats were randomly divided into 3 groups, sham-operated group, IR group and Lov group. The model of RIRI in IR and Lov groups was established by the clamping of the kidney artery for 60 minutes. Lovastatin was given 2mg/(kg·d) by oral gavage to the rats in the Lov group before clamping of the kidney artery. Ten rats in each group were executed at 4, 12, and 24 hours after reperfusion. The concentrations of serum creatinine (Cr) and urea nitrogen (BUN) were detected, and the expression of HSP70 and NF-KB in renal tissues was detected by immunohistochemistry. The pathologic changes of renal tissues were observed by microscope

  7. Effect of Baclicus lincheniformis on mRNA expression of Toll and HSP70 genes in Litopenaeus vannamei%地衣芽孢杆菌对凡纳滨对虾Toll和HSP70基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    曹煜成; 文国樑; 张华军; 张家松; 胡晓娟; 李卓佳


    目的 分析地衣芽孢杆菌对凡纳滨对虾Toll和HSP70基因表达的影响.方法 根据芽孢杆菌的使用量设置测试1组(LDG,用菌量2.0×103 CFU/mL),测试2组(HDG,用菌量1.0 × 104 CFU/mL)及未使用芽孢杆菌的对照组(CG),每7d施菌1次,实验持续24 d.每6d取样对虾肌肉和肝胰腺检测Toll基因和HSP70基因的mRNA相对表达量.结果 对虾肝胰腺Toll基因mRNA表达量在LDG组和HDG组分别较CG组提高了410.05%和78.31%,但在肌肉中CG组的表达量则分别较LDG组和HDG组提高了31.81%和8.43%.可见,芽孢杆菌在一定程度上可提高Toll基因mRNA在对虾肝胰腺的表达,但对其在肌肉中的表达则呈一定的限制作用.在肝胰腺HSP70基因的mRNA表达量,HDG组显著高于CG组和LDG组(P<0.05),CG组和LDG组的差异无统计学意义(P>0.05);在肌肉中LDG组和HDG组的HSP70基因mRNA表达分别较CG组提高了26.07%和26.46%,但实验组的组间差异无统计学意义(P>0.05).结论 在水体中施用芽孢杆菌104 CFU/mL和2.0×103 CFU/mL可分别提高对虾肝胰腺和肌肉HSP70基因的mRNA表达量.%Objective To study the effect of Baclicus on mRNA expression of Toll and HSP70 in Litopenaeus vannamei.Methods Three groups were established based on the concentrations of Baclicus used,including test group 1 (high dosage group,HDG),test group 2 (low dosage group,LDG) and control group (CG) ; the concentrations of Baclicus were 1.0 × 104 CFU/mL,2.0 × 103 CFU/mL and 0CFU/mL,respectively.Relative expression of Toll β-actin and HSP70 mRNA in hepatapancreas and muscle of L.vannamei were tested every 6 days,until the 24th day when this study ended.Results The relative Toll β-actin mRNA expressions in hepatapancreas in LDG and HDG were significantly higher than that of CG (P <0.01),increased by 410.05% and 78.31% respectively compared with CG,while those in muscle in were reduced by 31.81% and 8.43%,respectively.It indicated that B

  8. The immune responses and expression of metallothionein (MT) gene and heat shock protein 70 (HSP 70) in juvenile rockfish, Sebastes schlegelii, exposed to waterborne arsenic (As(3+)). (United States)

    Kim, Jun-Hwan; Kang, Ju-Chan


    Juvenile rockfish, Sebastes schlegelii (mean length 16.4±1.9cm, and mean weight 71.6±6.4g) were exposed for 20days with the different levels of waterborne arsenic concentration (0, 50, 100, 200 and 400μg/L). The plasma cortisol of S. schlegelii was significantly increased by the waterborne arsenit exposure. In the immune responses, the immunoglobulin M (Ig M) and lysozyme activity of S. schlegelii were significantly increased by the waterborne arsenic exposure. The acetylcholinesterase (AChE) activity of S. schlegelii was inhibited by the waterborne arsenic exposure. The substantial increases in the gene expression such as metallothionein (MT) and heat shock protein 70 (HSP 70) were observed by the waterborne arsenic exposure. The results demonstrated that waterborne arsenic exposure can induce the significant alterations in the immune responses and specific gene expression of S. schlegelii.

  9. 肝区微波照射后肝脏HSP70的表达及机制初探%Expression of Hepatic Heat Shock Protein 70 Induced by Microwave Irradiation on Liver Territory in Rabbits

    Institute of Scientific and Technical Information of China (English)

    童玲玲; 丁炳丽; 周筱艳; 李纯颍; 谢文彪; 袁秀琴; 谢红卫


    目的 采用20W、2450 MHz微波照射家兔肝区,观察照射过程中肝前区温度及肛温的变化,并于24小时后从家兔肝右中方叶前缘取组织用免疫组化方法检测HSP70的表达.方法 健康成年家兔13只,对其中4只在微波照射过程中肝前区温度及肛温的变化进行测定,其余9只分为两组,一组(n=4)取肝组织用免疫组化方法观察照射前肝HSP70的表达,另一组(n=5)进行微波预处理,24小时后观察肝脏HSP70的表达.结果 肝区微波照射过程中,家兔肝前区温度升高0.5~2.2℃,肛温升高0.5~0.7℃,24小时后肝细胞核内有HSP70的高表达,而预处理前动物肝细胞内无或仅有低水平HSP70的表达.结论 肝区微波照射过程中肝前区温度及肛温有轻度升高,24小时后肝细胞核内有HSP70的高表达.

  10. Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation. (United States)

    Schmidt, Béla Z; Watts, Rebecca J; Aridor, Meir; Frizzell, Raymond A


    Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation.

  11. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis. (United States)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan


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

  12. Free radical release and HSP70 expression in two human immune-relevant cell lines after exposure to 1800 MHz radiofrequency radiation. (United States)

    Lantow, M; Schuderer, J; Hartwig, C; Simkó, M


    The goal of this study was to investigate whether radiofrequency (RF) electromagnetic-field (EMF) exposure at 1800 MHz causes production of free radicals and/or expression of heat-shock proteins (HSP70) in human immune-relevant cell systems. Human Mono Mac 6 and K562 cells were used to examine free radical release after exposure to incubator control, sham, RF EMFs, PMA, LPS, heat (40 degrees C) or co-exposure conditions. Several signals were used: continuous-wave, several typical modulations of the Global System for Mobile Communications (GSM): GSM-non DTX (speaking only), GSM-DTX (hearing only), GSM-Talk (34% speaking and 66% hearing) at specific absorption rates (SARs) of 0.5, 1.0, 1.5 and 2.0 W/kg. Heat and PMA treatment induced a significant increase in superoxide radical anions and in ROS production in the Mono Mac 6 cells when compared to sham and/or incubator conditions. No significant differences in free radical production were detected after RF EMF exposure or in the respective controls, and no additional effects on superoxide radical anion production were detected after co-exposure to RF EMFs+PMA or RF EMFs+LPS. The GSM-DTX signal at 2 W/kg produced a significant difference in free radical production when the data were compared to sham because of the decreasing sham value. This difference disappeared when data were compared to the incubator controls. To determine the involvement of heat-shock proteins as a possible inhibitor of free radical production, we investigated the HSP70 expression level after different RF EMF exposures; no significant effects were detected.

  13. Energy sources and levels influenced on performance parameters, thyroid hormones, and HSP70 gene expression of broiler chickens under heat stress. (United States)

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


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

  14. Lead impact on nutrition, energy reserves, respiration and stress protein (hsp 70) level in Porcellio scaber (Isopoda) populations differently preconditioned in their habitats. (United States)

    Knigge, T; Köhler, H R


    The impact of lead on food consumption, energy metabolism and the stress protein (hsp 70) level was investigated in the woodlouse Porcellio scaber (Isopoda), a common representative of the saprophagous soil macrofauna. To examine possible acclimation or tolerance to lead in woodlice from a contaminated habitat, animals of two populations, one deriving from a lead-contaminated artillery range and one from an uncontaminated control stand, were exposed to a series of lead concentrations under otherwise constant laboratory conditions for a maximum of 80 days. The applied lead concentrations (at a maximum 7945 mg/kg food dry wt) did not have any significant quantitative effect on the food consumption of the isopods, although the population pre-exposed in the artillery range showed a tendency toward a higher food uptake than the control population. After 80 days of exposure, both populations showed an equal trend toward increasing their respiration as lead concentrations, that they had been fed on, were increased. Accordingly, the glycogen content of the body, in both populations, was elevated with increasing lead concentrations in the food. This effect was more pronounced in the pre-exposed isopod population than in the one from the control stand. The non-pre-exposed isopods showed a general tendency toward a lower protein content of their bodies than the pre-exposed ones, although no effect of the lead on this parameter could be statistically proven. The ability of the artillery range isopods to synthesise stress proteins (hsp 70) in response to lead contamination decreased at much lower lead concentrations in their food than in the non-pre-exposed control population, even though the artillery range isopods seemed to be equally or even slightly better equipped with energy storage products. Even though the better nutrient status of these animals might refer to some lead tolerance of the pre-exposed population, the stress protein data suggest that a metal

  15. Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico. (United States)

    Roy, Avijit; Stone, Andrew L; Shao, Jonathan; Otero-Colina, Gabriel; Wei, Gang; Choudhary, Nandlal; Achor, Diann; Levy, Laurene; Nakhla, Mark K; Hartung, John S; Schneider, William L; Brlansky, Ronald H


    Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

  16. 电针的不同波形对实验性大鼠急性痛风性关节炎滑膜组织中HSP70表达的影响%The Effect of Electro Acupuncture in Different Waveforms to HSP70 Expression in Rats of Acute Gouty Arthritis

    Institute of Scientific and Technical Information of China (English)

    金弘; 刘婷婷; 陈英华; 刘勇; 孙晓伟


    目的:明确不同波形电针治疗急性痛风性关节炎的疗效,探讨电针对实验性急性痛风性关节炎大鼠关节局部保护作用及其机制.方法:采用Coderre等经典造模方法建立急性痛风性关节炎大鼠模型.随机分成5组:空白组、模型组、电针疏波组、电针密波组、电针疏密波组.各组分成1天、3天、5天共3个时间点,选取“足三里”、“三阴交”穴进行电针治疗.以免疫组织化学技术观察病变关节滑膜组织中HSP70的表达变化,并进行统计学分析.结果:电针疏密波组能使急性痛风性关节炎大鼠滑膜内HSP-70蛋白表达显著增加(P<0.05或P<0.01),从而抑止滑膜组织炎症发生.结论:电针不同波形均能有效上调急性痛风性关节炎大鼠足踝关节滑膜内HSP70蛋白表达,从而减轻踝关节局部炎症反应,减轻关节损伤,且以电针疏密波为最佳.%Objective: To clear up the therapeutic effects of electroacupuncture in different waveforms to acute gouty arthritis, and reveal its mechanisms of partial protection for acute gouty arthritis. Methods: Coderre's Classical methods was used to establish the acute gouty arthritis model for rats. Wistar rats were divided ran- domly into five groups: blank group, model group, group of electroacupuncture in Shu - wave, group of elec-troacupuncture in dense -wave, group of electroacupuncture in Shu and dense wave. Each group was separated into 1 day ,3 day,and 5 day as subgroup for investigation. Select "Zusanli" , "Sanyinjiao" to be electroacupuncture points. To observe the changes of the expression of HSP70 in the diseased synovial tissue by using immuno-histochemical techniques and statistically analyzed the data. Results: Group of electroacupuncture in Shu and dense wave can significantly promote the expression of HSP - 70 protein in synovial tissues of acute gouty arthritis rats (P <0. 05 or P <0. 01 ) , thus restrains the occurrence of synovial tissue inflammation

  17. Expression of HSP70 family and HSP90 family in skeletal muscles of transport stressed pigs%运输应激猪骨骼肌中热应激蛋白HSP70和HSP90家族的表达

    Institute of Scientific and Technical Information of China (English)

    鲍恩东; Sultan,KR; 等


    To demonstrate the expression of heat shock protein s(HSP)in skeletal muscles and to estimate the relationship between expression of HSP and pork quality, using Western blot four kinds of HSP which belongs to HSP70 family and HSP90 family respectively in porcine skeletal muscle tissues were determined after a long distance transport.All four HSP,namely HSP70,HSP72,HSP86 and HSP90,were regularly detected in M.longissimus dorsi and M.gluteus medius of both the transported and the cont rol group.The expression of HSP in control pigs showed that HSPs have some indep endent physiological from stress response undetermined functions.after 6 h trans port,the induction of the HSP70 and HSP72 in skeletal muscle tissues was slightly elevated(P>0.05).However,expression of the HSP 86 and the HSP90,especially HSP90,markedly decreased(P<0.01)in skelet al muscles.The results are implied that the reduction tendency of HSP90 fa mily is relative to pork quality and may serve as a potential marker for stress response.%利用Western blot技术,检测长途运输试验猪骨骼肌(背最长肌和臀大肌)中分属于HSP70家族和HSP90家族的4种应激蛋白(HSP70,HSP 72,HSP86和HSP90)的表达。所有运输应激猪和对照猪肌肉组织中均检测到了上述4种HSP。对照猪组织中HSP的表达说明HSP除了在受应激的细胞内行使生理功能外,还具有独立于应激刺激应答以外的其它作用。6 h的长途运输后,HSP的表达量明显不同,HSP70和HSP72在肌肉组织中的表达虽然有一定的增加,但统计学分析差异不显著(P>0.05);而HSP86和HSP90在骨骼肌中的表达明显下降,尤以HS P90下降最为显著(P<0.01)。提示HSP90家族成员与肉品品质相关,可能作为判断应激损伤的指征。

  18. 预热适应对大鼠离心运动骨骼肌形态结构及HSP70表达的影H向%Effect of Heat Pretreat Adaptability on Skeletal Morphological Structure and the Expression of HSP70 in Rats after an Eccentric Exercise

    Institute of Scientific and Technical Information of China (English)

    朱洪竹; 肖国强


    目的:观察16天的预热适应对大鼠运动性骨骼肌损伤的影响;方法:成年大鼠80只,随机分为非预热适应组和预热适应组,各组又分为安静对照组、运动后即刻、运动后24h、运动后48 h组和运动后6d组.预热适应模型:热刺激30~35 min/天,共16天.末次热适应结束后,室温恢复24 h,除安静对照组外,其余大鼠均进行一次离心运动至动物宰杀.取腓肠肌,进行光镜观察和HSP70表达测试;结果:1)预热适应运动大鼠骨骼肌损伤较单纯运动大鼠相应的时间点明显减轻;2)预热适应运动大鼠运动后即刻和运动后48 hHSP70表达分别高于单纯运动后相对应时间点的值(P<0.05);结论:预热适应对大鼠运动性骨骼肌损伤有明显保护作用,其机理可能与长期(16天)的预热适应能上调HSP70蛋白表达有关.

  19. Stress inducible heat shock protein 70: a potent molecular and toxicological signature in arsenic exposed broiler chickens. (United States)

    Das, S; Pan, D; Bera, A K; Rana, T; Bandyopadhyay, S; De, S; Das, S K; Bhattacharya, D; Bandyopadhyay, S K


    This communication reports about heat shock protein response after arsenic exposure in broiler chickens in vivo and in vitro both. Splenocytes harvested in presence of sodium arsenite expressed Heat shock protein 70 (HSP 70) which could be identified on the basis of relative migration pattern and western blot analysis. Serum levels of HSP 70 in broiler chicken also increased after continuous feeding of sodium arsenite in drinking water. This particular observation may be attributed towards systematic inflammation, oxidative stress and hepatocellular injury. In vitro relative quantification of transcription level of HSP 70 revealed that splenocytes harvested in presence of sodium arsenite expressed (final concentration 3 and 7 μM/ml) more HSP 70 in comparison to cells harvested without sodium arsenite and the values were statistically significant (P < 0.001) when compared to untreated control. Collectively this result indicated that, HSP 70 level increased both in vivo and in vitro trials and may be used as potential molecular and toxicological biomarker.

  20. Genetic Transformation in Citrus

    Directory of Open Access Journals (Sweden)

    Dicle Donmez


    Full Text Available Citrus is one of the world’s important fruit crops. Recently, citrus molecular genetics and biotechnology work have been accelerated in the world. Genetic transformation, a biotechnological tool, allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. Citrus transformation has now been achieved in a number of laboratories by various methods. Agrobacterium tumefaciens is used mainly in citrus transformation studies. Particle bombardment, electroporation, A. rhizogenes, and a new method called RNA interference are used in citrus transformation studies in addition to A. tumefaciens. In this review, we illustrate how different gene transformation methods can be employed in different citrus species.

  1. 大鼠再生肝中hsbp1、hsf1、hsf2、hsp70表达水平改变的分析%ANALYSIS OF CHANGES ABOUT hsbp1, hsf1, hsf2 AND hsp70'S EXPRESSION LEVELS IN RAT'S REGENERATING LIVER

    Institute of Scientific and Technical Information of China (English)

    苏丽娟; 常翠芳; 韩鸿鹏; 马辉; 徐存拴



  2. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos. (United States)

    Maruyama, T; Ito, M; Honda, G


    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  3. Molecular cloning, characterization and functional analysis of a heat shock protein 70 gene in Cyclina sinensis. (United States)

    Ren, Yipeng; Pan, Heting; Yang, Ying; Pan, Baoping; Bu, Wenjun


    Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily and is involved in protecting organisms against various stressors. In the present study, we used RACE to clone a full-length Cyclina sinensis HSP70 cDNA termed CsHSP70. The full length of the CsHSP70 cDNA was 2308 bp, with a 5' untranslated region (UTR) of 42 bp, a 3' UTR of 268 bp, and an open reading frame (ORF) of 1998 bp encoding a polypeptide of 655 amino acids with an estimated molecular mass of 72.75 kDa and an estimated isoelectric point of 5.48. Quantitative real-time PCR was employed to analyze the tissue distribution and temporal expression of the CsHSP70 gene after bacterial challenge and cadmium (Cd) exposure. The CsHSP70 mRNA transcript was expressed ubiquitously in five examined tissues, with the highest expression in hemocytes (P < 0.05) and with the lowest expression in the hepatopancreas. Furthermore, the expression level of CsHSP70 in hemocytes at 3 h after Vibrio anguillarum challenge was extremely significantly up-regulated (P < 0.01). Moreover, the CsHSP70 transcript was up-regulated significantly following exposure to a safe Cd concentration (0.1 mg/L). Finally, after the CsHSP70 gene was silenced by RNA interference, the expression of the CsTLR13 and CsMyD88 genes were extremely significantly decreased (P < 0.01). The results indicated that CsHSP70 could play an important role in mediating the environmental stress and immune responses, and regulating TLR signaling pathway in C. sinensis.

  4. Phylogenetic Analysis of Peritrichs Inferred from Cytosolic Hsp70 Gene Partial Sequences%两种缘毛类纤毛虫胞质Hsp70基因序列的克隆及其系统发育分析

    Institute of Scientific and Technical Information of China (English)

    傅诚杰; 缪炜; 沈韫芬; 万明亮


    克隆得到2种缘毛类纤毛虫--钟形钟虫(Vorticella campanula)和螅状独缩虫(Carchesium polypinum)的胞质Hsp70基因部分序列,长度均为438 bp,编码146个氨基酸.以细菌为外类群,利用最大似然法和邻接法构建包括其他5种纤毛虫在内的共26个物种的Hsp70基因氨基酸序列系统发育树,其拓扑结构显示:V.campanula和C.polypinum聚在一起,并与另2种寡膜纲的嗜热四膜虫(Tetrahymena thermophila)及草履虫(Paramecium tetraurelia)聚为姊妹枝,提示了缘毛类纤毛虫为单系,且隶属于寡膜纲的系统发育地位.

  5. Expression of HSP70 in rat molars pulp tissue applied with orthodontic force%正畸力作用下大鼠牙髓组织中热休克蛋白70的表达及意义

    Institute of Scientific and Technical Information of China (English)

    苏士文; 赵紫婷; 程哲


    Objective To investigate the expression of heat shock protein 70 (Hsp70) in rat molars pulp applied with or-thodontic force. Methods The animal model for tooth movement was established, and the rats were sacrificed in batches on d1, 15 and 30. The maxil ary molars and periodontium specimens were obtained and the expression of Hsp70 was detected by im-munohistochemistry. Results The expression of Hsp 70 in control group and d1, d15, d30 orthodontic force groups were 4.02± 0.03 and 28.13 ±0.23, 10.02 ±0.08, 5.23 ±0.42, respectively. There was significant difference in Hsp 70 expression between control group and D1 orthodontic force group (P0.05). Conclusion During the orthodontic tooth movement, HSP70 might be one of main cytokines in pro-cess of reparative dentin mineralization.%目的:检测正畸力作用下大鼠牙髓组织中热休克蛋白70(Hsp70)的表达和分布,探讨其在正畸移动中的作用和机制。方法建立大鼠正畸牙移动模型,分别在受力1、15、30d处死,制备左侧上颌第一磨牙及其周围牙槽骨的石蜡标本,采用免疫组织化学法检测大鼠正畸牙移动过程中牙髓组织中Hsp70的表达情况。结果正常对照组、正畸加力1d组、正畸加力15d组和正畸加力30d组牙髓组织中Hsp70表达量分别为4.02±0.03、28.13±0.23、10.02±0.08、5.23±0.42,正畸加力1d组大白鼠牙髓组织中Hsp70表达量显著高于正常对照组(P<0.01),正畸加力15d组和正畸加力30d组与正常对照组的差异无统计学意义(P>0.05)。结论 Hsp70参与了正畸牙移动,并且保护了牙髓组织。

  6. Mekanisme Peningkatan Heat Shock Protein-70 pada Kanker Payudara Tikus yang Diradiasi, Pascapemberian Ekstrak Meniran (Phyllanthus niruri (MECHANISM OF INCREASING OF HSP-70 ON IRRADIATED RAT BREAST CANCER, DUE TO APPLICATION OF EXTRACT OF PHYLLANTHUS NIR

    Directory of Open Access Journals (Sweden)

    Bambang Soeprijanto


    Full Text Available The used of radiation as a cancer treatment is also proven giving the side effect of damaging thenormal tissue. The extract of Phyllanthus niruri L plant has already been known have an ability to modulatethe immune system. Heat Shock Protein 70 (HSP70 is a protein which can protect other proteins from anydamages. The transcription factor, such as Nuclear factor kappa-light-chain-enhancer of activated B cells(NfkB,is required for protein synthesis. This research was intended to analyze the mechanism, of the immunecompetent cells in expressing the HSP70,through the increase of Nf-kB, at the rat breast cancer tissueunder radiation, due to the application of the extract of P.niruri L plant. An experimental study wasperformed by using the randomized separate pre-test post test controlled group design. The female whiterat (Rattus norvegicus strain Sprague Dawleystrain, undergoing breast cancer due to the application ofcarcinogen materials 7,12-dimethylbenz(aantrasen(DMBA at 20 mg/kg.b.wt, and then the externalradiation at 6Gy given. The treatment group was given the aqueous extract of P.niruri L plant per oral at250 mg/kg b.wt.By immunohistochemistry and t-test study we observe a significant increases in thenumber of cells expressing Nf-kB (p<0.05 and a significant increases in the number of cells expressingHSP70 (p<0.05 at the treatment group. At the regression test, there found to be a stronger influence of NfkBto HSP70 at the treatment group. It is concluded that the mechanism of cell increase expressing theHSP70 at the rat breast cancer tissue under radiation due to the application of the aqueous extract ofP.niruri L plant per oral, is through the increase of cells expressing the Nf-kB.

  7. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement

    Directory of Open Access Journals (Sweden)

    Moghadamtousi SZ


    downregulation of Bax and upregulation of Hsp70 proteins after pretreatment. Collectively, the present results suggest that EEAM has a promising antiulcer potential, which could be attributed to its suppressive effect against oxidative damage and preservative effect toward gastric wall mucus. Keywords: Annona muricata, annonaceae, gastric injury, antioxidants, Hsp70/Bax

  8. Molecular Detection of Spiroplasma Citri Associated with Stubborn Disease in Citrus Orchards in Syria (United States)

    Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD) and can be transmitted from plant to plant by several species of phloem-feeding leafhoppers. CSD is an important disorder in certain warm and arid citrus-growing areas, and its agent has been recorded from several Med...

  9. Occurrence, molecular characterisation, and pathogenicity of Neoscytalidium dimidiatum on Citrus in Italy

    NARCIS (Netherlands)

    Polizzi, G.; Aiello, D.; Castello, I.; Vitale, A.; Groenewald, J.Z.; Crous, P.W.; Gentile, A.; La Malfa, S.


    During 2008 and 2009, a new disease was noticed in eastern Sicily (Italy) in two re-grafted citrus orchards (Citrus sinensis (L.) Osbeck) on sour orange rootstock. Symptoms consisted of blight of vigorously growing shoots and a sooty canker on shoots and rootstock trunks. A Scytalidium-like fungus w

  10. Occurrence, molecular characterisation, and pathogenicity of Neoscytalidium dimidiatum on citrus in Italy

    NARCIS (Netherlands)

    Polizzi, G.; Aiello, D.; Castello, I.; Vitale, A.; Groenewald, J.Z.; Crous, P.W.


    During 2008 and 2009, a new disease was noticed in eastern Sicily (Italy) in two re-grafted citrus orchards (Citrus sinensis (L.) Osbeck) on sour orange rootstock. Symptoms consisted of blight of vigorously growing shoots and a sooty canker on shoots and rootstock trunks. A Scytalidium-like fungus w

  11. Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis. (United States)

    Vaithinathan, S; Saradha, B; Mathur, P P


    Methoxychlor, an organochlorine pesticide, has been reported to induce abnormalities in male reproductive tract. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. We investigated whether treatment with methoxychlor would alter the levels of stress proteins, heat shock proteins (HSP), and clusterin (CLU), and oxidative stress-related parameters in the testis of adult male rats. Animals were exposed to a single dose of methoxychlor (50 mg/kg body weight) orally and were terminated at various time points (0, 3, 6, 12, 24, and 72 h) using anesthetic ether. The levels of HSP70, CLU, and the activities of superoxide dismutase (SOD), catalase, and lipid peroxidation levels were evaluated in a 10% testis homogenate. A sequential reduction in the activities of catalase and SOD with concomitant increase in the levels of thiobarbituric acid reactive substance (TBARS) was observed. These changes elicited by methoxychlor were very significant between 6-12 h of posttreatment. Immunoblot analysis of HSP revealed the expression of HSP72, an inducible form of HSP, at certain time points (3-24 h) following exposure to methoxychlor. Similarly, the levels of secretory CLU (sCLU) were also found to be elevated between 3-24 h of treatment. The present data demonstrate methoxychlor-elicited increase in the levels of inducible HSP72 and sCLU, which could be a part of protective mechanism mounted to reduce cellular oxidative damage.

  12. CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system. (United States)

    Cabral Miranda, Felipe; Adão-Novaes, Juliana; Hauswirth, William W; Linden, Rafael; Petrs-Silva, Hilda; Chiarini, Luciana B


    Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress.

  13. Bos indicus cattle possess greater basal concentrations of HSP27, alpha B-crystallin, and HSP70 in skeletal muscle in vivo compared with cattle. (United States)

    Mullins, C R; Zerby, H N; Fitzpatrick, L A; Parker, A J


    The objectives of the present study were to evaluate the basal concentrations of heat shock proteins (HSP) between and cattle and to determine if HSP basal concentrations change as an animal matures. A total of 40 cattle were used in a 2 × 2 factorial design to evaluate the effects of genotype and age (heifers and mature cows) on basal concentrations of heat shock protein 27 (HSP27), α B-crystallin (Cryab), and heat shock protein 70 (HSP70). Each experimental group of 10 animals was sampled on a separate day over a period of 4 wk during July 2014. A muscle sample was collected from the longissimus thoracis (LT) and concentrations of HSP were quantified using ELISA. There were no significant differences in HSP concentration for the interaction between age and genotype or for age alone. cattle had greater ( cattle. The results of this study show that basal in vivo HSP concentrations differ between and cattle. However, further studies are needed to investigate the relationship between HSP concentrations and meat tenderness with respect to genotypes to see if HSP concentrations account for at least some variability in tenderness differences.

  14. Versatile members of the DNAJ family show Hsp70 dependent anti-aggregation activity on RING1 mutant parkin C289G (United States)

    Kakkar, Vaishali; Kuiper, E. F. Elsiena; Pandey, Abhinav; Braakman, Ineke; Kampinga, Harm H.


    Parkinson’s disease is one of the most common neurodegenerative disorders and several mutations in different genes have been identified to contribute to the disease. A loss of function parkin RING1 domain mutant (C289G) is associated with autosomal-recessive juvenile-onset Parkinsonism (AR-JP) and displays altered solubility and sequesters into aggregates. Single overexpression of almost each individual member of the Hsp40 (DNAJ) family of chaperones efficiently reduces parkin C289G aggregation and requires interaction with and activity of endogenously expressed Hsp70 s. For DNAJB6 and DNAJB8, potent suppressors of aggregation of polyglutamine proteins for which they rely mainly on an S/T-rich region, it was found that the S/T-rich region was dispensable for suppression of parkin C289G aggregation. Our data implies that different disease-causing proteins pose different challenges to the protein homeostasis system and that DNAJB6 and DNAJB8 are highly versatile members of the DNAJ protein family with multiple partially non-overlapping modes of action with respect to handling disease-causing proteins, making them interesting potential therapeutic targets. PMID:27713507

  15. Effect of Benzo[a]pyrene or/and Heat on Levels of HSP70 and HSP90 β in Mice Brain%苯并[a]芘和高温及其联合作用对小鼠脑组织HSP70和HSP90 β水平的影响

    Institute of Scientific and Technical Information of China (English)

    涂白杰; 邬堂春; 贺涵贞


    目的研究苯并[a]芘(BaP)、高温及其联合作用对小鼠脑组织热应激蛋白70(HSP70)与热应激蛋白90β(HSP90 β)表达的影响.方法将56只昆明种小鼠随机分为7组(每组8只), 即空白对照组、溶剂对照组、低剂量BaP单独染毒组、高剂量BaP单独染毒组、单独高温处理组、高温处理+低剂量BaP染毒组、高温处理+高剂量BaP染毒组.低、高剂量BaP染毒分别给予BaP 0.5mg/kg和5mg/kg,用植物油作溶剂,每周4次腹腔注射,溶剂对照组用植物油作平行处理,空白对照组不做处理.高温处理为每周4次,每次41.5~42.0℃处理2 h.实验8周后取各组小鼠脑组织制作混合匀浆,Westernblot法检测HSP70和HSP 90 β水平.结果 HSP70在高温单独作用、低剂量BaP单独作用、高温与低剂量BaP联合作用下表达明显升高.HSP90 β仅在高温与低剂量BaP联合作用及高温与高剂量BaP联合作用下表达升高.结论不同的热应激蛋白在不同条件下对高温、BaP染毒等有害因素对脑组织的损害起保护和标志作用.

  16. 电击死大鼠心脏超微结构及HSP70、HIF-1α表达变化%Cardiac Ultrastructure and Changes of HSP70 and HIF-1α Expression in Electric Shock Death Rats

    Institute of Scientific and Technical Information of China (English)

    张国忠; 李瑞利; 冯国伟; 毕海涛; 王松军; 丛斌; 左敏


    目的:观察电击死大鼠心脏超微结构及热休克蛋白70(heat shock protein 70,HSP70)、低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的表达变化,探讨其是否能作为电击死法医学鉴定的依据。方法将72只SD大鼠随机分为电击死组、死后电击组和对照组。通过透射电镜观察电击死大鼠心肌超微结构的改变,并采用免疫组织化学技术观测心肌HSP70和HIF-1α的表达变化。结果电击大鼠心肌细胞肌原纤维断裂,线粒体嵴和膜溶解消失,Z线、M线排列紊乱。 HSP70和HIF-1α在电击死组呈强阳性表达,与死后电击组、对照组比较差异有统计学意义(P<0.05)。结论 HSP70和HIF-1α在电击死组表达明显上调,有望作为电击死的诊断参考指标。%Objective T o observe cardiac ultrastructure and the expression of heat shock protein 70 (H SP70) and hypoxia inducible factor-1α (H IF-1α) in electric shock death rats and to explore the application of these indexes as the basis of m edical identification in electric shock death. Methods Seventy-tw o SD rats w ere random ly divided into electric shock death group, postm ortem electric shock group and the control group. T he changes of m yocardial ultrastructure w ere observed by transm ission electron m icro-scope, and the expressions of m yocardial H SP70 and H IF-1α w ere observed by im m unohistochem ical technology. Results M yocardial m yofibril fracture, m itochondrial cristae and m em brane dissolution, and disordered arrangem ent of Z lines and M lines w ere observed in electric shock rats. H SP70 and H IF-1αw ere strong positive expressions in the electric shock death group, significantly com pared w ith the con-trol and postm ortem electric shock groups (P<0.05). Conclusion T he expressions of H SP70 and H IF-1αw ere obviously increased in electric shock death group, w hich m ay be used as the diagnostic indicator of electric shock

  17. Effect of Ganoderma lucidum spores on cytochrome C, mitochondrial Ca2+, HSP70 and BDNF in brain of epilepsy rats%灵芝孢子粉对癫痫大鼠脑组织细胞色素C、线粒体钙、HSP70和BDNF的影响

    Institute of Scientific and Technical Information of China (English)

    王淑秋; 王淑湘; 李晓捷; 姜志梅; 秦晓玉; 韩玉泽; 刘蕾; 刘君星; 王芳芳; 梁衍峰


    目的:研究灵芝孢子粉对癫痫大鼠脑组织超氧化物歧化酶(SOD)、丙二醛(MDA)、总抗氧化能力(T-AOC)、细胞色素C、热休克蛋白70(HSP70)、线粒体Ca2+和脑源性神经营养因子(BDNF)的影响,探讨灵芝孢子粉对癫痫大鼠脑细胞的保护作用机制.方法:用亚惊厥剂量的戊四氮(PTZ)复制癫痫慢性模型,采用火焰原子吸收法测定脑组织线粒体Ca2+的含量,分光光度计比色法测定SOD的活性、MDA和细胞色素C含量,免疫组织化学方法检测HSP70和BDNF的表达情况.结果:与癫痫模型组相比,灵芝孢子粉组线粒体内细胞色素C、线粒体Ca2+的含量和HSP70的表达显著增高,而胞浆内细胞色素C含量则明显降低;脑组织SOD和T-AOC的活性明显增加,而MDA含量明显降低.大脑皮质和海马区BDNF阳性细胞数癫痫模型组明显高于正常对照组 (P<0.05);灵芝孢子粉组皮质和海马区BDNF阳性神经元数目进一步增多,与癫痫模型组相比,差异显著(P<0.05).结论:灵芝孢子粉能明显降低癫痫发作对脑细胞线粒体造成的损伤,其作用机制可能是通过清除自由基、增强脑组织的抗氧化能力,从而减轻自由基对线粒体膜的损伤作用,恢复线粒体的能量代谢,减轻脑细胞的损伤与凋亡.%AIM : To investigate the effects of Ganoderma lucidum spores on superoxide dismutase( SOD ),malondialdehyde( MDA ),total antioxidative capacity( T - AOC ), cytochrome C, heat - shock protein 70 ( HSP70 ), mitochondrial Ca2+ and hrain - derived neurotrophic factor( BDNF ) in the brain tissues of epilepsy rats.METHODS : The rat hronic epilepsy model was hy intraperitoneal injection of pentetrazole( PTZ ) at a subconvulsant dose ( 32 mg/kg ).Flame atomic absorption method was used to detect the content of mitochondrial Ca2 + , and spectrophotometer colorimetry was used to measure SOD activity,MDA content,T - AOC and cytochrome C levels in rat brain tissues.HSP70 and BDNF were de termined

  18. The Hsp70-like StkA functions between T4P and Dif signaling proteins as a negative regulator of exopolysaccharide in Myxococcus xanthus

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    Pamela L. Moak


    Full Text Available Myxococcus xanthus displays a form of surface motility known as social (S gliding. It is mediated by the type IV pilus (T4P and requires the exopolysaccharide (EPS to function. It is clear that T4P retraction powers S motility. EPS on a neighboring cell or deposited on a gliding surface is proposed to anchor the distal end of a pilus and trigger T4P retraction at its proximal end. Inversely, T4P has been shown to regulate EPS production upstream of the Dif signaling pathway. Here we describe the isolation of two Tn insertions at the stk locus which had been known to play roles in cellular cohesion and formation of cell groups. An insertion in stkA (MXAN_3474 was identified based on its ability to restore EPS to a pilA deletion mutant. The stkA encodes a DnaK or Hsp70 homolog and it is upstream of stkB (MXAN_3475 and stkC (MXAN_3476. A stkB insertion was identified in a separate genetic screen because it eliminated EPS production of an EPS+ parental strain. Our results with in-frame deletions of these three stk genes indicated that the stkA mutant produced increased level of EPS while stkB and stkC mutants produced less EPS relative to the wild type. S motility and developmental aggregation were affected by deletions of stkA and stkB but only minimally by the deletion of stkC. Genetic epistasis indicated that StkA functions downstream of T4P but upstream of the Dif proteins as a negative regulator of EPS production in M. xanthus.

  19. The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa. (United States)

    Yeste, Marc; Estrada, Efrén; Rivera Del Álamo, Maria-Montserat; Bonet, Sergi; Rigau, Teresa; Rodríguez-Gil, Joan-Enric


    Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h-30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (Psperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance.

  20. 海豚链球菌感染对不同品系罗非鱼血液生化指标和肝脏HSP70 mRNA表达的影响%Studies on blood biochemical indices and expression of hepatic HSP70 mRNA of different tilapia strains artificially challenged with Streptococcus iniae

    Institute of Scientific and Technical Information of China (English)

    强俊; 杨弘; 王辉; 徐跑; 柒壮林; 何杰


    In order to establish the superiority, four different tilapia strains (GIFT tilapia, New GIFT tilapia, Egypt Nile tilapia and Red tilapia) were compared in the study. At day 100 tilapias in the treatment group were intraperitoneally injected with Streptococcus iniae suspension (2.95×108/mL) to evaluate disease resistance of four different tilapia strains against S. iniae in terms of the serum biochemical parameters and hepatic HSP70 mRNA expression before and after infection. Another set of twenty fish taken from each tank was challenged with S. iniae using the same method as above, and the cumulative mortality of 4 strains was recorded at different times post infection. The results showed that GIFT tilapia and New GIFT tilapia were more sensitive to S. iniae at 96 h after infection, cumulative mortalities were 36.67% and 38.33% respectively. Egypt Nile tilapia was less sensitive to pathogen and no death occurred during the experiment. The levels of serum cortisol (COR) and glucose (GLU) and expression levels of HSP70 mRNA in GIFT tilapia, New GIFT tilapia and Red tilapia strains were highly significant(P<0.05), and the activities of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST)and lysozyme (LSZ) also increased, while serum alkaline phosphatase (AKP), triglyceride and cholesterol levels were significantly lower than those prior to infection (P<0.05). By utilizing the energy from resolved hepatin and lipid, the synthesis of hepatic HSP70 mRNA and levels of LSZ and globulin in Egypt Nile tilapia were improved, and thus the non-specific immunity strengthened. It is suggested that in the course of tilapia breeding, effective combination of the disease resistance with growth be conducted so as to guarantee sustainable development of the tilapia industry.%以吉富罗非鱼、新吉富罗非鱼、埃及尼罗罗非鱼和红罗非鱼为研究对象,饲养100 d后,进行海豚链球菌(2.95×108 CFU/mL)感染试验,分析攻毒前后各品

  1. The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa.

    Directory of Open Access Journals (Sweden)

    Marc Yeste

    Full Text Available Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT of 4 h-30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (P<0.05 increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance.

  2. Influence of HSP70 on function and energy metabolism of mitochondria in intestinal epithelial cells after hypoxia/reoxygenation%热休克蛋白70对肠上皮细胞缺氧/复氧后线粒体功能及能量代谢的影响

    Institute of Scientific and Technical Information of China (English)

    袁志强; 李晓鲁; 彭毅志; 王裴; 黄跃生; 杨宗城


    目的 了解Ad-热休克蛋白70(HSP70)对缺氧/复氧损伤后肠上皮细胞线粒体功能及能量代谢的影响.方法 取肠上皮细胞株IEC-6分别转染Ad-HSP70腺病毒载体和空腺病毒载体,蛋白质印迹法观察HSP70的表达.取IEC-6细胞分为正常对照组(不作任何处理)、缺氧/复氧组(给予缺氧/复氧处理)、Ad-HSP70转染组(转染Ad-HSP70腺病毒载体后,给予缺氧/复氧处理).采用噻唑蓝比色法检测细胞内线粒体脱氧酶的活性,高效液相色谱分析法测定细胞能量代谢.结果 与转染空腺病毒载体相比,转染Ad-HSP70腺病毒载体可显著增加细胞HSP70的表达.缺氧/复氧组细胞内线粒体脱氢酶的活性明显低于正常对照组(P<0.01),Ad-HSP70转染组该指标明显高于缺氧/复氧组(P<0.01).缺氧/复氧组细胞内腺苷三磷酸含量较正常对照组显著下降,而腺苷二磷酸、腺苷-磷酸含量显著升高;Ad-HSP70转染组细胞能量指标与正常对照组相似(P>0.05),较缺氧/复氧组有明显改善(P<0.05或P<0.01).缺氧/复氧组细胞能荷为0.615±0.060,明显低于正常对照组(0.748±0.012,P<0.01)、Ad-HSP70转染组(0.736±0.028,P<0.01).结论 Ad-HSP70腺病毒载体转染肠上皮细胞可诱导HSP70表达增加,显著提高细胞缺氧/复氧后胞内腺苷三磷酸的含量及细胞能荷,保护线粒体整体功能,提示线粒体是HSP70保护肠上皮细胞缺氧/复氧损伤的主要靶细胞器之一.%Objective To investigate the effect of recombinant adenovirus-mediated heat shock pro-tein 70(HSP70)on energy metabolism of mitochondria in intestinal epithelial cells(IEC-6)after hypoxia/reoxygenation injury. Methods IEC-6 cells were transfected with HSP70 recombinant adenovirus vectors (Ad-HSP70)and empty adenovirus vectors.The expression of HSP70 protein was detected by Western blot-ting.Cultured IEC-6 cells were divided into:control group(without treatment),hypoxia/reoxygenation group(with challenge of

  3. Effects of nicorandil, glutamine, metoprolol used in single and combination on antiapoptosis ability of cardiocytes and expression of HSP70 against myocardial ischemia/reperfusion injury in rats%尼可地尔、谷氨酰胺、美托洛尔单独及合用对大鼠心肌缺血/再灌注损伤后心肌细胞抗凋亡和HSP70的影响

    Institute of Scientific and Technical Information of China (English)

    陈文华; 孙畅; 张颖; 吴艳娜; 温克; 康毅; 娄建石


    目的:研究尼可地尔( nicorandil,Nic)、谷氨酰胺( glu-tamine,Glu)、美托洛尔( metoprolol,Met)三药联用对大鼠心肌缺血/再灌注损伤后心肌细胞抗凋亡能力和保护性蛋白HSP70表达的影响。方法将健康裔 Wistar大鼠随机分为7组:①假手术( Sham )组,只实施冠状动脉左前降支( left anterior descending,LAD)穿线;② I/R组,实施LAD 30 min缺血/120 min再灌注程序( ischemia/reperfusion,I/R);③Nic组;榆 Glu组;虞 Met组;愚三药联用( NGM)组;舆三药联用低剂量( NGML)组。药物预处理组则在执行I/R程序前30 min腹腔注射Nic、Glu和Met,三药的初始给药剂量均为1 mg·kg-1。 TUNEL(terminal dexynucleotidyl transferase-medi-ated dUTP nick end labeling)法检测心肌细胞凋亡情况,通过凋亡指数衡量心肌细胞凋亡率;Western blot法检测凋亡相关蛋白Bcl-2、Bax以及保护性蛋白热休克蛋白70(heat shock protein 70,HSP 70)的表达情况。结果 I/R组中,缺血区的细胞凋亡率较高(36.9%依10.3%),细胞凋亡相关蛋白表达Bcl-2/Bax值较低(0.14依0.08);与I/R组相比,药物预处理组的细胞凋亡率明显降低(P<0.01),凋亡相关蛋白表达Bcl-2/Bax值明显升高(P<0.01);与Sham组相比,I/R组的HSP70表达明显增加( P<0.01);与I/R组相比,药物预处理组的HSP70表达明显增加( P<0.01);与药物单用组相比,3药联用组的HSP70表达更高( P<0.01)。结论对于大鼠在体I/R损伤,三药联用组与药物单用组相比细胞凋亡率明显降低,凋亡相关蛋白Bcl-2、Bax的表达有所改善;保护性蛋白HSP70的表达增加。%Aim To investigate the protective effects of nicorandil, glutamine and metoprolol used in single and combination on the antiapoptosis ability of cardio-cytes and the expression of HSP70 after myocardial is-chemia/reperfusion injury in rats. Methods Male Wistar rats were divided randomly into seven groups:( 1 ) Sham group:in which the coronary artery was not


    Institute of Scientific and Technical Information of China (English)

    田照辉; 徐绍刚; 王巍; 胡红霞; 马国庆


    The effect of acute thermal stress on HSP1Q mRNA expression, physiology and nonspecific immunity were investigated in Siberian sturgeon. In the experiment, ten fish were transferred froml7.5℃ to 27.5℃ water, then data were measured at lh and 3h. Fish in 17.5℃ water were taken as Oh group. Respiratory rate increased from 80-90 breaths/min to 210 breaths/min, then decreased to 180 breaths/min after 0.5h. Among the three tissues including gill, spleen and brain, the HSP70 mRNA expression in gill rose most quickly after lh stress, and was about 1.63 fold compared with the Oh group (P<0.05), then remained this level to 3h; The HSP10 mRNA expression in spleen and brain had nearly no change in lh thermal stress, then rose quickly from lh to 3h in the two tissues, especially in brain at 3h rose most quickly to about 1.77 fold compared with the lh group (P<0.05). Compared with the Oh group, serum cortisol increased quickly and was 5.14 fold (PO.05) at lh, then decreased quickly to 2.1 fold at 3h. Spleen macrophage respiratory burst achieved the maximum (P<0.05) at lh, then reduced to the Oh group level. Serum complement C3 increased at lh and decreased significantly at 3h (P<0.05). Serum lysozyme activity firstly elevated then decreased but had no significant differences. Serum SOD activity decreased when thermal stress time prolonged, and achieved the minimum at 3h (P<0.05). Serum MDA content decreased when thermal stress time prolonged and had no significant difference. The results showed that lh acute thermal stress promoted the nonspecific immunity and tolerance, but 3h acute thermal stress decreased it.%为研究西伯利亚鲟(Acipenser baerii)对急性热应激的抗逆机理,将体质量为(155.47±19.50) g的鱼从17.5 ℃迅速转至27.5 ℃水中,在1h和3h取样测定HSP70 mRNA表达变化、血清皮质醇和非特异性免疫指标.结果显示:急性热应激时鳃、脾和脑的HSP70 mRNA表达量升高,具有组织特异性,热应激1h时

  5. The Interactions of TPR Domains of Ppp5c/TTC16 with Hsp70/90 and Their Influence on the Cell Cycle%Ppp5c和TTC16基因的TPR结构域与Hsp70、Hsp90蛋白的相互作用及对细胞周期的影响

    Institute of Scientific and Technical Information of China (English)

    刘德康; 李蕾; 陈霞; 唐超; 李建民


    目的:研究Ppp5c及TTC16基因的TPR (tetratricopeptide repeat)结构域短片段和Hsp70及Hsp90家族蛋白的相互做用,及其过表达时细胞周期的影响.方法:通过生物信息学的分析及PCR的方法,克隆Ppp5c及TTC16基因的TPR结构域以及HSPAIA、HsP90AA1的全长基因,并连入酵母双杂交载体,通过ClonTech的酵母双杂交实验体系研究蛋白和蛋白之问的相互作用.把Ppp5c及TIC16基因的TPR结构域克隆入真核表达栽体,构架稳定表达Ppp5c及TTC16基因的TPR结构域的MCF-7细胞系,并通过流式细胞实验观察细胞周期.结果:Ppp5c及TTC16基因的TPR结构域能与HSPAIA或HsP90AAI发生相互作用.Ppp5c及TTC16基因的TPR结构域在MCF-7中的过表达能严重影响细胞周期,引起细胞凋亡和S期阻滞.结论:本实验初步揭示了不同蛋白的TPR结构域在与Hsp70及Hsp90蛋白的相互作用性质的异同点以及其过表达对细胞周期的影响,为全面理解TPR结构域的功能、PPP5c以及TTC16蛋白在细胞内的功能奠定了前期实验基础.%Objective: To investigate the interactions between the TPR (tetratricopeptide repeat) domains of Ppp5c/TTC16 and protein, of Hsp70/Hsp90, to study the effect of the overexpression of TPR domains of Ppp5c/TTC16 on cell cycle in MCF-7 cell lines. Methods: After being analyzed by online informatics tools, the TPR domains of Ppp5c/TTC16 and full-length of HSPA1A/HSP90AA1 genes were respectively sub-cloned into two separate vectors of the yeast two-hybrid system provided by ClonTech to confirm the protein-protein interactions. The TPR domains of Ppp5c/TTC16 were sub-cloned into eukaryotic expression vectors which were stably transfected into MCF-7 cell-lines, whose cell cycle was then examined by flow cytometry. Results: The TPR domains of Ppp5c/TTC16 interacted with proteins of HSPA1A/HSP90AA1. A large amount of apoptosis was detected when the TPR domains of Ppp5c over-expressed in MCF-7 cell line, and the cell cycle was

  6. Application of Bioinformatics in the Study of HSP70 Recombinant Vaccine of Echinococcus Granulosus%生物信息学技术在细粒棘球蚴热休克蛋白70重组疫苗研究中的应用

    Institute of Scientific and Technical Information of China (English)

    丁淑琴; 刘宏鹏; 张爱君; 张彩芳; 赵巍


    目的 通过基因序列分析,寻找细粒棘球蚴(Echinococcus granulosus,Eg)热休克蛋白基因(Heat shock pm-tein7,HSP70),为包虫病防治筛选新的候选疫苗抗原.方法 ①登录 GenBank 公共数据库,检索 EgHSP70目的基因序列.②对克隆基因进行测序,结果输入DNAStax、BioSun分析软件和互联网 SWISS-MODEI 数据库,对重组 EgHSP70 的特性、抗原表位及构象进行预测分析.结果 ①获得了具有完整开放阅读框的 EgHSP70核苷酸序列.②DNAStar分析软件推算氨基酸序列结果显示,EgHSP70由133个理论氨基酸组成,分子量约为14.53kDa.DNAStar、BioSun分析软件确定了重组 EgHSP70 可能的抗原表位.与其他动物热休克蛋白70氨基酸的同源性分析结果显示EgHSP70具有高度的保守性.结论 生物信息学技术在 EgHSP70 重组疫苗研究中有一定的理论和应用价值.

  7. A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elsa Perrody

    Full Text Available The universally conserved J-domain proteins (JDPs are obligate cochaperone partners of the Hsp70 (DnaK chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32 facilitates RB43 bacteriophage proliferation is discussed.

  8. Detection and molecular identification protocols for Phyllosticta citricarpa from citrus matter

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    Mariette Truter


    Full Text Available Strict quarantine measures for the export of South African citrus fruit to European and US markets require the development of sensitive and accurate detection methods for the pathogen Phyllosticta citricarpa – a fungus causing citrus black spot disease. Because of the presence of other, non-pathogenic Phyllosticta species, rapid and accurate verification of the Phyllosticta species present on exported citrus fruit is important to producers, exporters and regulatory authorities to prevent unnecessary losses. We have analysed over 800 samples collected over 7 years and have compared sample preparation and detection protocols applied in different environments: nurseries, production systems including phytosanitary inspections in orchards, pack houses and export terminals in order to compile protocols for the detection of P. citricarpa. Standard procedures of sample preparation and DNA extraction were adapted to suit diverse inoculum sources. Low pathogen numbers in symptomless green leaves, for example, obliged the use of a wet-dry enrichment technique constituting the stimulation of fungal growth for easier detection. Physical maceration was adapted for sturdy material using liquid nitrogen or bead beating. The use of a two-step polymerase chain reaction (PCR with nested primers significantly increased both the sensitivity and the specificity of the PCR performed on soil samples, overcoming problems with relatively impure DNA extracts and low pathogen numbers. The assays have proven to be highly consistent, thereby providing a reliable, reproducible and highly sensitive detection and diagnostic service to the southern African citrus industries in order to sustain market access.

  9. Genetic Structure and Molecular Variability Analysis of Citrus sudden death-associated virus Isolates from Infected Plants Grown in Brazil

    Directory of Open Access Journals (Sweden)

    Emilyn Emy Matsumura


    Full Text Available Citrus sudden death-associated virus (CSDaV is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR, the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5’ end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies.

  10. Genetic Structure and Molecular Variability Analysis of Citrus sudden death-associated virus Isolates from Infected Plants Grown in Brazil (United States)

    Matsumura, Emilyn Emy; Coletta Filho, Helvécio Della; de Oliveira Dorta, Silvia; Nouri, Shahideh; Machado, Marcos Antonio


    Citrus sudden death-associated virus (CSDaV) is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD) disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR), the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π) was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5’ end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies. PMID:27999249

  11. Molecular characterization of Wolbachia strains associated with the invasive Asian citrus psyllid Diaphorina citri in Brazil. (United States)

    Guidolin, A S; Cônsoli, F L


    Wolbachia is a symbiont intensively studied due to its ability to interfere with their host's reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing" (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host-symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.

  12. 溶氧反馈-补料分批技术高密度培养基因重组MAGE1/HSP70/MAGE3工程菌%Preparation of gene recombinant MAGE1/HSP70/MAGE3 by high cell density culture of E.coli.with DO fed-back control of nutrient feeding

    Institute of Scientific and Technical Information of China (English)

    胡沛臻; 温江田; 路凡; 王孝功; 李侠; 司少艳; 葛伟; 黄杨; 张秀敏; 隋延仿


    目的 建立人黑色素瘤抗原MAGE1/HSP70/MAGE3融合蛋白基因重组工程菌E.coli.BL21/pET-MHM的高密度发酵工艺.方法 以摇瓶发酵结果为基础,扩大至NBS Bioflo Ⅳ 20 L发酵罐发酵,利用溶氧反馈-补料分批培养技术,对影响工程菌生长及目的蛋白表达的因素如发酵培养基、活化时间、诱导浓度及时间、pH值及分批补加营养物质等进行优化.结果 保持培养过程中40%的溶解氧,采用半合成培养基、活化至对数生长期时0.2 mmol/L IPTG诱导5 h以及以甘油为碳源连续流加补料的条件发酵,连续3批重复发酵,最终菌密度D(600)均达45~50时,菌体量可提高至70 g/L以上,目的蛋白的表达量占菌体蛋白总量的38%以上.结论 确定了周期短、产率高且稳定可靠的发酵工艺.

  13. Effects of dendritic cells transfected with full length wild type p53 and modified by GC-HSP70 on immune response%胃癌抗原肽Hsp70复合物对转染全长野生型p53的树突状细胞免疫功能的影响

    Institute of Scientific and Technical Information of China (English)

    孙华文; 唐启彬; 邹声泉


    目的应用联合修饰的树突状细胞(DC)在体外诱导高效而特异的抗胃癌免疫效应.方法先将全长野生型p53导入脂质体内并转染小鼠骨髓来源的DC,然后用胃癌抗原肽-HSP70复合物等因素修饰已转染全长野生型p53的DC(wt-p53 DC),检测这种DC诱导小鼠脾脏淋巴细胞特异的细胞毒性T淋巴细胞(CTLs)的能力,分泌细胞因子功能,以及表面分子表达的高低.结果 Western blot检测转染小鼠p53cDNA的BMDC及其培养上清中均可以检测到p53表达;细胞因子含量明显增加(P<0.05),而的其他组的细胞因子含量与对照相比,无显著变化;经流式细胞仪(FACS)检测wt-p53DC表面高表达B7-1、B7-2、MCH-Ⅰ、MCH-Ⅱ;脾淋巴细胞经刺激后,能够特异性地杀伤胃癌细胞,杀伤率为91.6%.结论全长野生型p53基因转染+抗原肽联合修饰DC能诱导小鼠细胞毒性T淋巴细胞的特异性,显著提高DC的抗原提呈功能.

  14. Influence of Dietary Energy Level on Hepatic70-kDa Heat Shock Protein Expression in Broiler Chickens Submitted to Acute Heat Stress Influência do Nível de Energia da Dieta sobre a Expressão Hepática de Hsp70-kDa em Frangos Submetidos ao Estresse Calórico Agudo

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    JE Gabriel


    Full Text Available This experiment was carried out to study the effect of dietary energy on the colonic temperature and hepatic Hsp70 content in broiler chicken at room temperature and after heat stress conditions. Broiler chickens were reared up to 51 days of life, and fed diets containing high (HE -13,186 kJ ME/kg or low (LE -12,139 kJ ME/kg energy. At 21 and 51 days of age, the colonic temperature was measured at room temperature and liver samples were obtained for Hsp70 quantification by Western blotting analysis. It was also investigated at these ages the time course response of colonic temperature and hepatic Hsp70 level during heat stress (35º C/5 h. The data showed that at early age, at room temperature, colonic temperature or hepatic Hsp70 levels were not affected by dietary energy, but at 51 days of life low energy fed broilers had lower Hsp70 concentration in the liver. During heat stress, the increase in both colonic temperature and hepatic Hsp70 concentration were significantly less in high energy fed birds. The findings of this study suggest that hepatic Hsp70 synthesis is affected by dietary energy, and that broiler chicken fed high-energy diet can change the thermoresistance during acute heat stress.Este trabalho foi desenvolvido com o objetivo de pesquisar o efeito da energia da dieta sobre a temperatura do cólon e concentração de proteína de choque térmico (Hsp70 de frangos à temperatura ambiente, bem como durante o estresse calórico agudo. Os frangos foram criados até 51 dias de idade e alimentados com dietas contendo nível de energia alto (13.186 kJ EM/kg ou baixo (12.139 kJ EM/kg. No 21º e 51º dias de idade, a temperatura do cólon foi medida e amostras de fígado foram obtidas para quantificação da Hsp70 através da análise por Western Blotting.. Nessas mesmas idades, a resposta das aves ao estresse calórico agudo (37º C/5 h foi avaliada (temperatura colón e Hsp70 no fígado. Os resultados mostraram que aos 21 dias de idade

  15. H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment. (United States)

    Gu, H; Smith, M W; Phelps, P C; Berezesky, I K; Merriman, R L; Boder, G B; Trump, B F


    The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

  16. Echinococcus granulosus 2 heat shock protein 70 gene: construction of recombinant plasmid, prokaryotic expression, purification and characterization%细粒棘球蚴2HSP70重组质粒的构建、原核表达、纯化和初步鉴定

    Institute of Scientific and Technical Information of China (English)

    丁淑琴; 王洁; 王淑静; 张焱; 赵巍


    目的 构建细粒棘球蚴(Echinococcus granulosus,EG)2热休克蛋白70(heat shock protein,2HSP70)表达重组质粒,原核表达及纯化重组蛋白,并对重组蛋白进行免疫学特性的初步鉴定.方法 从EG2HSP70/pGEM-T/JM109质粒中分离EG2HSP70目的基因,将此片段重组入表达载体pGEX-6P-1后转化入大肠杆菌BL21,鉴定后以IPTG诱导表达,通过SDS-PAGE检测其表达水平,用亲和层析纯化并分离重组蛋白,利用Western blot来鉴定重组蛋白的免疫学特性.结果 酶切鉴定及测序分析表明,成功构建细粒棘球蚴pGEX-6P-1/EG2HSP70重组质粒;IPTG诱导表达后,融合蛋白占菌体总蛋白的39%,占裂解物上清总蛋白的70.4%,表明重组的EG2HSP70基因能在BL21中高效表达,表达的蛋白大部分以可溶性状态存在.亲和层析法纯化重组蛋白EG2HSP70,通过Western blot证实该重组蛋白能被细粒棘球蚴囊壁免疫兔血清识别.结论 成功构建细粒棘球蚴中国大陆株pGEX-6P-1/EG2HSP70重组质粒,重组的EG2HSP70能够高效表达,表达的EG2HSP70具有免疫学活性.

  17. Genetic diversity and phylogenetic analysis of Citrus (L from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA

    Directory of Open Access Journals (Sweden)

    Marlykynti Hynniewta


    Full Text Available The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L. species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply

  18. Resveratrol modulates intestinal morphology and HSP70/90, NF-κB and EGF expression in the jejunal mucosa of black-boned chickens on exposure to circular heat stress. (United States)

    Liu, Lili; Fu, Chenxing; Yan, Mingli; Xie, Hongbing; Li, Si; Yu, Qifang; He, Shaoping; He, Jianhua


    The aim of this study was to investigate whether supplementation with resveratrol could alleviate intestinal injuries and to explore how resveratrol regulates heat shock protein (HSP)70, HSP90, nuclear factor kappa B (NF-κB) and epidermal growth factor (EGF) expression in the jejunal mucosa of black-boned chickens under circular heat stress. A total of 300 black-boned chicks of 42-d-old were randomly assigned to five treatment groups. The positive control chickens were kept in a normal-temperature (NT, 24 ± 2 °C) chamber and fed with a basal diet. The other four groups were kept in a circular high-temperature (HT, 37 ± 2 °C) chamber for 8 h and fed a basal diet supplemented with 0, 200, 400, or 600 mg per kg of resveratrol for 15 days. The results showed that the heat-stress responses damaged the villus structures of the jejunum and ileum, resulting in shortened intestinal villi, deepened crypts, and a reduced villus height to crypt depth (V/C) ratio and decreased the numbers of goblet cells and lymphocytes. Heat stress also caused increased mRNA and protein expression of HSP70, HSP90 and NF-κB, and reduced EGF in the jejunal mucosa. Dietary supplementation with 400 mg per kg of resveratrol improved the villus morphology, increased the numbers of goblet cells and lymphocytes, attenuated the mRNA overexpression of HSP70, HSP90 and NF-κB on the 6th, 10th and 15th day of heat stress (P stress, and increased EGF expression from the lamina propria toward the epithelial cells of the villi. These results suggest that dietary resveratrol offers a potential nutritional strategy to improve the intestinal morphology and alleviate jejunum mucosa injuries by modulating the mRNA and protein expression of HSPs, and the epithelial growth factor and transcription factor in black-boned chickens subjected to circular heat stress.

  19. Molecular characterization of a citrus yellow vein clearing virus strain from China. (United States)

    Zhen, Song; Kurth, Elizabeth G; Peremyslov, Valera V; Changyong, Zhou; Dolja, Valerian V


    The complete nucleotide sequence of an isolate of citrus yellow vein clearing virus from Yunnan, China (CYVCV-RL), was determined following whole-genome amplification by RT-PCR. The CYVCV-RL genome was 7529 nt in length, excluding the 3' poly (A) tail, and contained six open reading frames (ORFs), resembling that of viruses belonging to the genus Mandarivirus in the family Alphaflexiviridae. Sequence analysis showed that the CYVCV-RL shared the greatest nucleotide sequence identity with the CYVCV-Y1 (JX040635) isolate from Turkey for the whole genome (97.1%), 5' UTR (98.7%), 3' UTR (100.0%), and each of six ORFs (96.5% to 97.8%), suggesting that there is apparent genetic stability among CYVCV isolates of different geographic origin.

  20. Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant (United States)

    Romero, Paco; Rodrigo, María J.; Alférez, Fernando; Ballester, Ana-Rosa; González-Candelas, Luis; Zacarías, Lorenzo; Lafuente, María T.


    Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and -independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage. PMID:22315241

  1. Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W. (United States)

    Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J; Primo-Millo, Eduardo; Forner-Giner, M Ángeles


    This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3

  2. Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis. (United States)

    Shonhai, Addmore; Maier, Alexander G; Przyborski, Jude M; Blatch, Gregory L


    Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.

  3. Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots (United States)

    Xie, Rangjin; Zhang, Jin; Ma, Yanyan; Pan, Xiaoting; Dong, Cuicui; Pang, Shaoping; He, Shaolan; Deng, Lie; Yi, Shilai; Zheng, Yongqiang; Lv, Qiang


    Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. ‘MAPK cascade’), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway’), reactive oxygen species (ROS) metabolic process (e.g. ‘hydrogen peroxide catabolic process’) and transcription factors (e.g., ‘MYB, ZFP and bZIP’) were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment. PMID:28165059

  4. The destructive citrus pathogen, 'Candidatus Liberibacter asiaticus' encodes a functional flagellin characteristic of a pathogen-associated molecular pattern.

    Directory of Open Access Journals (Sweden)

    Huasong Zou

    Full Text Available Huanglongbing (HLB is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las, retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla of 452 amino acids that contains a conserved 22 amino acid domain (flg22 at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of Fla(Las. Agrobacterium-mediated transient expression in planta revealed that Fla(Las induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine and 39 (aspartate of Fla(Las were essential for callose induction. The synthetic flg22(Las peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that Fla(Las acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.

  5. 脑室内低温对家兔急性大脑中动脉缺血模型HSP70表达的影响%The effects of cerebral ventricle low temperature Oil the expression of heat shock protein 70 in middle cerebral artery occlusion (MCAO) rabbits

    Institute of Scientific and Technical Information of China (English)

    姚冰; 姚长义


    目的 观察脑室内低温对家兔急性大脑中动脉缺血模型热休克蛋白70((Heat shock protein 70,HSP70)表达的影响.方法 家兔16只,随机分为2组:对照组(常温生理盐水)、低温组(22℃低温生理盐水)各8只,应用线栓法建立大脑中动脉闭塞(MCA0)模型,进行脑室内穿刺低温液体灌注亚低温干预,24 h后采用SABC方法检测各组脑组织HSP70表达,TUNEL方法检测凋亡细胞.结果 低温组的TUNEL染色阳性细胞数和HSP70染色阳性细胞数明显低于及多于常温组,有显著性差异(P<0.05).结论 通过脑室内低温治疗,可以减少缺血区域凋亡细胞,并增加HSP70细胞的表达.同时改善了动物的神经系统症状.

  6. Effects of astragalus polysaccharides (APS) injection on the expression of heat stress proteins 70 (hsp70) gene in Oreochromis niloticus%注射黄芪多糖对吉富罗非鱼热应激蛋白基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    汤菊芬; 吴灶和; 简纪常; 鲁义善; 王蓓


    将黄芪多糖(APS)用无菌生理盐水配制成2 mg/mL(低剂量)、20 mg/ mL(高剂量)两种针剂,腹腔注射吉富罗非鱼(Oreochromis niloticus).分别在注射后0h、24h、48 h、72 h和96 h采样提取吉富罗非鱼鳃、头肾、肝脏和脾脏组织中的总RNA,并反转录成cDNA,利用Real-time PCR方法对不同组织中的热应激蛋白(hsp70)基因表达进行定量分析.结果显示,在24h时,高剂量组吉富罗非鱼鳃、头肾和肝脏三个组织中hsp70基因的表达量显著高于同期对照组(P<0.05),之后表达量快速降低,低剂量组hsp70基因表达量仅在脾脏中有上调.实验表明,APS可通过诱导吉富罗非鱼hsp70基因表达来保护免疫细胞免受应激损伤,从而增强鱼体抵抗病原菌的能力.%Astragalus polysaccharides (APS) was dissolved in sterile saline at the doses of 2 mg/mL (low dose), 20 mg/mL (high dose) to inject Oreochromis niloticus intraperitoneally, and the effects of APS on gene expression of hsp70 in gill, head kidney, liver and spleen of 0. Niloticus were determined using quantitative real-time polymerase chain reac tion at 24 h, 48 h, 72 h and 96 h after intraperitoneal injection. The results revealed that the gene expression level of hsp70 in gill, head kidney and liver of high dose group increased significantly than those of control group (P <0. 05) at 24 h, but decreased quickly afterwards. The gene expression of hsplO was up-regulated only in the spleen in low dose group (P <0.05) at 24 h. These results indicated that APS could protect immune cell from heat injury and enhance the fish immunity against infection of bacterial pathogen by inducing the expression of hsp70 of 0. Niloticus.

  7. 快速眼动睡眠剥夺后大鼠皮质及海马 HSP70表达的研究%Rapid eye movement sleep deprivation on expression of heat shock protein 70 in rat cortex and hippocampus

    Institute of Scientific and Technical Information of China (English)

    黄建欧; 田国红; 赵忠新; 贺斌; 黄流清


    目的探讨不同时间的快速眼动(REM)睡眠剥夺对大鼠皮质及海马各区的热休克蛋白70(HSP70)表达的影响及意义.方法60只Wistar大鼠,随机分为睡眠剥夺组(SD)、环境对照组(TC)和空白对照组(CC).其中SD组又分为1d、3d、5d、7d 4个时点.用改良多平台睡眠剥夺法进行REM睡眠剥夺,运用免疫组织化学方法观察REM睡眠剥夺后大鼠额叶、顶叶皮质及海马各区HSP70表达的分布规律及时空变化;同时结合蛋白质免疫印记(Western Blot)实验对额叶皮质及全海马HSP70蛋白作了选择性的半定量分析.结果REM睡眠剥夺后1 d脑内HSP70表达一过性增强,以后逐渐下降.Western Blot实验印证了这一结果.结论REM睡眠剥夺能够引起大鼠皮质及海马神经元内HSP70表达增强,可能是一种自身稳定调节的保护机制.

  8. The effects of Tween 80 in combination with 42℃ hyperthermia on proliferation and protein expression of human ovarian cancer cell line SKOV-3%Tween 80联合42℃温热对人卵巢癌SKOV-3细胞增殖及HSP70蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    蔡素霞; 田晓予; 米建强; 段文艳; 冯磊; 王一佳


    目的: 观察Tween 80联合42℃温热对人卵巢癌SKOV-3细胞热耐受和热休克蛋白(heat shock protein,HSP70)表达的影响.方法:采用MTT法测定Tween 80联合42℃温热作用后,SKOV-3细胞的生长抑制率改变;采用免疫细胞化学法测定Tween 80联合42℃温热作用后SKOV-3细胞的HSP70表达情况.结果:Tween 80联合42℃温热组的SKOV-3细胞生长抑制率较单纯作用组明显升高(P<0.05);Tween 80联合42℃重复温热组的细胞增殖率较单纯42℃重复温热组明显下降(P<0.05);不同体积分数(0.025%、0.05%、0.075%和0.1%)的Tween 80联合42℃温热组与单纯温热组相比,SKOV-3细胞的HSP70表达均下降,并且随着Tween 80浓度增加,HSP70的表达逐渐下降.结论:Tween 80联合42℃温热对人卵巢癌SKOV-3细胞的生长抑制可能有相加或协同作用,Tween 80可抑制加热后细胞的HSP70表达和热耐受性产生.

  9. La proteína de estrés calórico Hsp70 funciona como un indicador de adaptación de los bovinos a las zonas áridas

    Directory of Open Access Journals (Sweden)

    Bañuelos-Valenzuela, Rómulo, Sánchez-Rodríguez, Sergio H.


    Full Text Available El propósito del presente estudio fue seleccionar genotipos de ganado bovino que estén mas adaptados al estrés calórico en las zonas áridas, a través de la expresión de las proteínas de choque calórico (Hsp, las cuales, son constituyentes normales de las células y su síntesis se incrementa con la exposición a varias formas de estrés. Nuestro objetivo fue establecer, si la expresión de las Hsp de los linfocitos del ganado bovino explotado en la región, muestra diferencias raciales en respuesta al estés calórico. De los bovinos, se obtuvieron linfocitos de sangre periférica por gradientes de Ficoll-histopaque, después se cultivaron en medio RPMI a temperaturas de 38, 40, 42 y 44°C, durante 4 horas, posteriormente se lisaron, se obtuvieron extractos que se caracterizaron por PAGE-SDS 10%, se transfirieron a papel de nitrocelulosa y se probaron con anticuerpos monoclonales anti-Hsp 25, 60,70 y 90. Se encontró: El calor es un agente estresante en los linfocitos del ganado bovino explotado en la región, cuya respuesta es a través de la expresión de la proteína Hsp70. La cantidad de proteína Hsp70 expresada en los linfocitos esta en función al estrés calórico recibido. El nivel de adaptación de un organismo ante un estresor, esta en función a la cantidad de Hsp expresada, por lo tanto, las razas bovinas más adaptadas a la región en orden decreciente es: Holstein-Australiano, Criollo, Pardo Suizo, Holstein y Limousin.

  10. Molecular Characterization of Vitellogenin and Its Receptor Genes from Citrus Red Mite, Panonychus citri (McGregor

    Directory of Open Access Journals (Sweden)

    Rui Zhong


    Full Text Available The production and uptake of yolk protein play an important role in the reproduction of all oviparous organisms. Vitellogenin (Vg is the precursor of vitellin (Vn, which is the major egg storage protein, and vitellogenin receptor (VgR is a necessary protein for the uptake of Vg into developing oocytes. In this paper, we characterize the full-length Vg and VgR, PcVg1 and PcVgR, respectively, of the citrus red mite Panonychus citri (McGregor. The PcVg1 cDNA is 5748 nucleotides (nt with a 5553-nt open reading frame (ORF coding for 1851 amino acids (aa, and the PcVgR is 6090 nt, containing an intact ORF of 5673 nt coding an expected protein of 1891 aa. The PcVg1 aa sequence shows a typical GLCG domain and several K/RXXR cleavage sites, and PcVgR comprises two ligand-binding domains, two epidermal growth factor (EGF-like regions containing YWTD motifs, a transmembrane domain, and a cytoplasmic domain. An analysis of the aa sequences and phylogenetics implied that both genes were genetically distinct from those of ticks and insects. The transcriptional profiles determined by real-time quantitative PCR in different developmental stages showed that both genes present the same expressional tendencies in eggs, larvae, nymphs, and adults. This suggested that the biosynthesis and uptake of PcVg occurs coordinately. The strong reproductive capacity of P. citri has been hypothesized as an important factor in its resistance; consequently, understanding the molecular mechanisms regulating Vg and VgR are fundamental for mite control.

  11. Chaperoning Proteins for Destruction: Diverse Roles of Hsp70 Chaperones and their Co-Chaperones in Targeting Misfolded Proteins to the Proteasome

    Directory of Open Access Journals (Sweden)

    Ayala Shiber


    Full Text Available Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases.

  12. Genes and longevity: a genetic-demographic approach reveals sex- and age-specific gene effects not shown by the case-control approach (APOE and HSP70.1 loci). (United States)

    Dato, S; Carotenuto, L; De Benedictis, G


    Association analyses between gene variability and human longevity carried out by comparing gene frequencies between population samples of different ages (case/control design) may provide information on genes and pathways playing a role in modulating survival at old ages. However, by dealing with cross-sectional data, the gene-frequency (GF) approach ignores cohort effects in population mortality changes. The genetic-demographic (GD) approach adds demographic information to genetic data and allows the estimation of hazard rates and survival functions for candidate alleles and genotypes. Thus mortality changes in the cohort to which the cross-sectional sample belongs are taken into account. In this work, we applied the GD method to a dataset relevant to two genes, APOE and HSP70.1, previously shown to be related to longevity by the GF method. We show that the GD method reveals sex- and age-specific allelic effects not shown by the GF analysis. In addition, we provide an algorithm for the implementation of a non-parametric GD analysis.

  13. Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice. (United States)

    Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen


    Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

  14. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone. (United States)

    Modisakeng, Keoagile W; Jiwaji, Meesbah; Pesce, Eva-Rachele; Robert, Jacques; Amemiya, Chris T; Dorrington, Rosemary A; Blatch, Gregory L


    Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

  15. Intratumor injection of recombinant attenuated salmonella carrying Mycobacterium tuberculosis heat shock protein 70 and herpes simplex virus thymidine kinase genes to suppress murine melanoma growth%mtHSP70/HSV-tk重组沙门菌抗小鼠黑色素瘤的作用

    Institute of Scientific and Technical Information of China (English)

    曾曙光; 刘启才; 王素文; 彭细毛; 章锦才; 张积仁


    目的 研究携带结核杆菌热休克蛋白70(mtHSP70)、人单纯疱疹病毒一胸苷激酶(HSV-TK)双基因的重组沙门菌瘤内注射抗小鼠黑色素瘤的抑瘤效应.方法 构建重组沙门菌SL7207/PCMV-mtHSP70-IRES-TK,建立小鼠黑色素瘤动物模型,瘤内注射重组沙门菌观察其抑瘤效应、荷瘤鼠的生存期并进行安全性评估.结果 瘤内注射重组沙门菌,实验组抑瘤率显著高于对照组,延长荷瘤鼠生存期,重组菌治疗后荷瘤小鼠的生存状态良好,无腹泻,治疗期间体质量无明显改变.结论 减毒沙门菌携带的mtHSP70/HSV-TK双基因真核共表达质粒瘤内注射对B16肿瘤细胞具有显著抑制作用.%Objective To study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes. Methods Plasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group. Results In vivo, the mtHSP70/ HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10' CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly. Conclusion Results of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth.

  16. Study on the relationship between HSP70 expression and DNA damage in human embryonic lung(HEL)cells by benzo(a)pyrene metabolites%苯并(a)芘代谢产物作用下人胚肺细胞热应激蛋白70表达与DNA损伤

    Institute of Scientific and Technical Information of China (English)

    高雅娟; 陈胜; 肖成峰; 王瑞波; 贺涵贞; 邬堂春


    Objective To investigate the relationship between HSP70 expression and DNA damage in human embryonic lung(HEL) cells treated by benzo(a)pyrene(BaP) metabolites in vitro. Methods HEL cells of unheated group were stimulated by 0, 10,50,100,200 μ moL/L BaP for 3 h incubation(induced by S9-mix in vitro). HEL cells of heated group were treated at 41 ℃ for 1 h,recovered 2 h at 37 ℃ ,then were stimulated by BaP at the same conditions as the untreated group. The DNA damage and HSP70 expression were determined by single cell gel assay and Western blot respectively. Results DNA damage in HEL cells induced by BaP was detectable at 50 μmol/L in untreated group, while DNA damage in heated group was detectable at10 μmol/L. The level of HSP70 expression decreased,but DNA damage of HEL cells reduced at the highest dose(200 μmol/L BaP ) compared with the control. Compared with the untreated group, the number of cells with “+ +” level DNA damage reduced( P 0.05). Conclusion The roles of HSP70 in the protection from DNA damage induced by BaP are not significant, which may be related to the inhibition of HSP70 expression by BaP.%目的探讨苯并(a)芘(BaP)代谢产物作用下,人胚肺细胞HSP70表达与DNA损伤的关系。方法选用正常人胚肺(HEL)二倍体细胞,进行如下处理:一般处理组,以0、10、50、100、200μmol/L BaP染毒3 h(体外经大鼠肝S9-mix活化诱导),单细胞凝胶电泳技术检测DNA损伤情况;热应激组,细胞预热应激(41℃1 h,37℃2 h),再以同样浓度的BaP染毒3 h,采用Westem blot和单细胞凝胶电泳技术检测HSP70表达与DNA损伤情况。结果一般处理组BaP在50μmo