豚鼠庆大霉素耳中毒后HSP70 mRNA的表达

为探讨热休克蛋白(heatshockprotein,HSP)70mRNA在庆大霉素(gentamicin,GM)耳中毒中的意义,本实验选用耳廓反射灵敏的健康白色红目豚鼠(200~250g)20只,雌雄不拘,随机分成两组,每组10只。实验组动物每日腹腔注射GM100mg/kg;对照组动物每日腹腔注射与GM等量的生理盐水2.5ml/kg。两组动物混合饲养,均连续用药10d。在用药前1天和停药后第1天进行听脑干反应(auditorybrainstemresponse,ABR)测试。各组豚鼠在行第二次ABR检测后,应用原位杂交及图像分析技术观察GM耳中毒后HSP70mRNA在豚鼠耳蜗中表达。结果显示:实验组耳蜗ABR阈值明显高于对照组,有显著性差异(P<0.01);实验组豚鼠耳蜗血管纹、螺旋韧带、螺旋神经节细胞HSP70mRNA表达呈强阳性,其平均灰度值较正常对照组明显减小(P<0.001),即GM能显著增强耳蜗HSP70mRNA的表达。结果提示,GM中毒后,动物可能通过增加HSP70mRNA在耳蜗的表达,起保护听力的作用。     

Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells

Phenylarsine oxide (PAO)forms a stable ring complex with vicinal dithiols that can be reversedwith 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or2-mercaptoethanol (2-ME). PAO at 2 µM or higher inhibited heat shockprotein 70 (HSP70) induction within minutes in cultured guinea piggastric mucosal cells exposed to heat (43°C) for 30 min. PAO did notaffect the nuclear translocation and phosphorylation of heat shockfactor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading tothe block of expression of HSP70 mRNA and accumulation of HSP70 in thecells. These inhibitions were completely reversed with 2 µM DMP butnot with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactionsbetween PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx)reversed the inhibition of the binding activity of HSF1 in whole cellextracts prepared from PAO-treated, heat-stressed cells. Our resultssuggest that PAO may react with vicinal-containing molecules includingTrx and specifically block the interaction between HSF1 and HSE.

     

Amphibian oocytes release heat shock protein A during spawning: evidence for a role in fertilization

Heat shock proteins A (HSPAs, previously known as HSP70s) are widely distributed proteins originally linked with heat shock but now associated with several normal cellular functions. We recently found indirect evidence suggesting a role for HSPAs in sperm-oocyte interaction in the amphibian Bufo arenarum. In the present study our aim was to study its expression, subcellular distribution, and role during fertilization. By Western blot analysis using two different antibodies we detected HSPAs present in B. arenarum oocytes in the absence of any stress. We performed two-dimensional electrophoresis and detected two isoforms with isoelectric points of 5.25 and 5.45. We studied its subcellular distribution isolating total membranes, cytosol, and plasma membranes. HSPAs were present in all of these fractions. We confirmed these results by immunofluorescence microscopy and also found that the HSPA signal was present in the vitelline envelope. To further test this, we performed Western blot analysis in isolated vitelline envelopes and in egg water (diffusible material from deposited oocytes). HSPAs were present in these two fractions. Moreover, human recombinant his-tagged HSPA (HSPA1A) was able to specifically bind to sperm in vitro (midpiece) and enhance sperm membrane integrity. In vitro fertilization assays in the presence of anti-HSPA polyclonal antibodies showed diminished fertilization scores at low sperm concentrations (10(5) cells per milliliter). Our results suggest that HSPAs are present in intracellular and extracellular structures of nonstressed B. arenarum oocytes and participates in fertilization by and that their release during spawning plays a role in sperm membrane integrity.     

Structural changes in chromatin of heat shock protein 70 gene of Drosophila during transcription

Using "protein-image" hybridization technique combined with various crosslinking methods, for formaldehyde-prefixed nuclei we have analysed changes induced by activation in the chromatin structure of HSP-70 genes. From the crosslinking data it follows that chromatin of actively transcribed genes undergoes some structural rearrangements resulting in certain weakening of the contacts between DNA and the globular parts of histones so that the histones remain bound to DNA through their N-terminal regions. In addition, there have been found two specific regions with a reduced content of histones: the 5'-promoter of HSP-70 gene and a region distanced by approximately 1 k.b. from the 3'-end of the HSP-70 gene.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Expression and sulfogalactolipid binding specificity of the recombinant testis-specific cognate heat shock protein 70

Immunofluorescent studies with anti-2A antisera, raised specifically against a synthetic C-terminal peptide of native murine P70, the testes-specific cognate heat shock protein 70, demonstrated that the rat homologue of P70 is expressed on the surface of testicular cells. The murine hsp 70.2 gene, encoding P70, was cloned and expressed in Escherichia coli. The recombinant P70 (rP70) protein with a 6Xhistidine affinity tag at its amino terminus was purified from E. coli via nickel affinity column chromatography. Monoclonal anti-hsp70 antisera and anti-2A antisera cross-reacted with purified rP70. Binding of rP70 was specific for sulfogalactosylceramide (SGC) and sulfogalactosyglycerolipid (SGG). Binding was not inhibited by the sugar, galactose 3′ sulfate, nor was binding observed to desulfated derivatives of SGC and SGG, to other negatively charged lipids or other sulfated lipids. Furthermore, rP70 bound to an SGC-column and was eluted only at high salt in combination with high pH. These results show rP70 to possess a specific sulfatide binding site. Since the biochemical properties and immunoreactivity of rP70 are indistinguishable from native P70 and SLIP1 (testicular sulfoglycolipid immobilized protein 1) rP70 can be employed to examine the role of hsp70-mediated sulfatide binding in fertilization. Abbreviations: PAGE, polyacrylamide gel electrophoresis; IPTG, isopropylthio-β-D-galactoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactoside; km, kanamycin; amp, ampicillin; kDa, kilodaltons; kb, kilobases; BHI, brain heart infusion; OPD, O-phenylenediamine dichloride peroxidase substrate; RT, room temperature; h, hours This revised version was published online in November 2006 with corrections to the Cover Date.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

Endothelial cells downregulate expression of the 70 kDa heat shock protein during hypoxia

Hsp70 is induced by hypoxia in most mammalian cell types and contributes to their ability to survive hypoxic episodes. However, little is known about Hsp70 expression in the hypoxia-tolerant endothelial cells (ECs). We investigated the effect of hypoxia on Hsp70 in human microvascular endothelial HMEC-1 cells. Reduction of pO(2) to 2.5% of normal for 20 h stimulated lactate production and the activity of glycolytic enzymes. This metabolic adaptation to hypoxia was accompanied by a remarkable reduction of Hsp70 on the protein level and on the mRNA level. Approximately 12 h after the hypoxic period Hsp70 expression reached pre-hypoxia levels again. Since ECs are adapted to the low oxygen tension of the vasculature they are confronted with a supraphysiological oxygen level during in vitro culture. We suppose that the high Hsp70 under these conditions reflects a stress response which disappears at the more physiological reduced oxygen tension during hypoxia.     

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.     

HSP70-inducible hNIS-IRES-eGFP reporter imaging: response to heat shock

A retroviral vector pQHSP70/hNIS-IRES-eGFP (pQHNIG70) was constructed containing the hNIS-IRES-eGFP dual-reporter genes under the control of an inducible human heat shock protein (HSP)70 promoter and RG2-pQHSP70/hNIS-IRES-eGFP (RG2-pQHNIG70) transduced cells were generated. Heat-induced expression of both reporter genes in RG2-pQHNIG70 cells was validated by enhanced green fluorescent protein (eGFP) fluorescence-activated cell sorter, in vitro radiotracer assays, and immunoblot and immunocytochemistry. A 2.2- to 6.1-fold ((131)I(-)), a 6.1- to 14.4-fold ((99m)TcO(4)(-)), and a 5.1- to 39-fold (fluorescence) increase above baseline was observed in response to graded hyperthermia (39-43 degrees C). Increases in eGFP fluorescence and radiotracer uptake were first noted at 6 hours, reached a maximum at 24 hours, and fell toward baseline at 72 hours. A stable ratio of radiotracer uptake to eGFP fluorescence and to heat shock protein (HSP)70 protein was demonstrated over a wide range of expression levels, induced by different levels of heating. We also demonstrate that the local application of heat on RG2-pQHNIG70 xenografts can effectively induce hNIS and eGFP gene expression in vivo and that this expression can be efficiently visualized by fluorescence, scintigraphic, and micro-positron emission tomography imaging. Endogenous HSP70 protein and reporter expression was confirmed by postmortem tissue evaluations (immunoblot and immunohistochemistry). The pQHNIG70 reporter system can be used to study stress and drug responses in transduced cells and tissues.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.