Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

<abstract>Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1±3.2 and 243.0±21.6, respectively, while those after thawing were 23.2±2.5 and 105.7±28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

Hepatitis C virus inhibitor synergism suggests multistepinteractions between heat-shock protein 90 and hepatitis Cvirus replication

AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.

肉鸡组织脏器的急性热应激损伤及HSP_(90)的表达

将 30日龄AA肉鸡在 ( 4 0± 1)℃高温中分别持续 2、 3、 5和 10h ,定期剖杀 ,进行临床血液病理学、组织病理学和免疫组织化学检测。结果表明 :在热应激 2～ 5h内 ,受试鸡外周血液中肌酸激酶活性持续升高 (P <0 0 1) ;当热应激持续到 10h时其活性出现降低 ,但仍明显高于对照组 (P <0 0 1)。受试鸡的组织脏器在应激前期损伤较严重 ,热应激 2～ 5h内 ,各主要脏器组织如心肌纤维、肝细胞、肾小管上皮细胞出现颗粒变性和脂肪变性 ,甚至坏死 ;而后期组织细胞的损伤则无明显坏死。提示在热应激持续一定时间后 ,机体可通过自身调节逐渐获得对高热的耐受能力。免疫组化结果显示 ,HSP90 广泛分布在热应激受试鸡脏器组织细胞的胞浆和胞核中 ,尤其在组织的小动脉、毛细血管内皮细胞内呈现强表达。提示血管壁中HSP90 可能通过血管的舒缩而影响组织器官的血液供应。

HSP70和HSP90α在人膀胱癌中的表达及意义

目的 探讨热休克蛋白70(heat- shockprotein70,HSP70)和热休克蛋白90α(heat shockprotein90α,HSP90α)在 膀胱癌中的表达及临床意义。方法 应用免疫组化技术检测HSP70、HSP90α和增殖细胞核抗原(proliferatingcellnuclearan tigen,PCNA)在50例膀胱癌组织和14例正常膀胱粘膜组织的表达。结果 膀胱癌中HSP70、HSP90α阳性表达率分别为 56%(28 50)和66%(33 50)。HSP70和HSP90α在膀胱癌病理分级、临床分期、生存率等方面具有显著性差异(P<0.05)。 HSP70、HSP90α和PCNA在膀胱癌中表达呈正相关。结论 HSP70、HSP90α与膀胱癌细胞分化,肿瘤的浸润深度有关,在膀 胱癌发生和发展中起重要作用。HSP70、HSP90α可以作为判断膀胱癌预后的新指标。

难治性肾病综合征GR、HSP90与中医证型关系研究

难治性肾病综合征（ refractory nephritic syndrome,RNS）是指符合肾病综合征的临床诊断,在应用激素规范治疗后病情不能缓解,或频繁复发,或激素依赖的病例[1]。因蛋白尿持续存在,肾脏损害进行性加重,易进展至终末期肾病。研究表明[2]经10年随访,RNS者有50%发生肾衰竭,激素敏感者仅有3%发生肾衰竭。中医药治疗能拮抗激素的副作用,提高机体对激素的敏感性及增强疗效、减少蛋白尿,减少复发等作用。本研究对 RNS 不同中医证型患者检测其外周血单个核细胞（ pe-ripheral blood mononuclear cell, PBMC ）糖皮质激素受体α、β（GRα、GRβ）、热休克蛋白90（heat-shock protein 90,HSP90）的表达差异,探讨其与证候的相关性,对指导中医诊治RNS、揭示中医药作用机制及治疗靶点有一定意义,现报道如下。

HSP90表达与牛精子抗冻性研究进展

热应激蛋白(HSPs)是生物体抵抗温度升高、氧化刺激和缺氧等环境变化时广泛合成的一组蛋白。论文简述了冷冻-解冻后精子蛋白质含量变化的研究进展以及热应激蛋白HSP90表达量对牛冷冻精液质量影响的机理,为提高牛精液冷冻品质提供理论参考。

二苯乙烯苷对APP/PS1/Tau三转基因小鼠痴呆模型CK1、HSP90、 PP5的影响

目的观察二苯乙烯苷(TSG)对淀粉样前体蛋白/早老蛋白-1/tau蛋白(APP/PS1/Tau)三转基因小鼠痴呆模型脑组织中酪蛋白激酶(CK)1、磷酸丝氨酰/磷酸苏氨酰蛋白磷酸酶(PP)5、热休克蛋白(HSP)90的表达影响。方法8月龄APP/PS1/Tau三转基因小鼠45只,随机分为模型组、阳性药对照组(石杉碱甲0.15 mg/kg)、TSG低、中、高剂量组(TSG 0.033、0.100、0.300 g/kg),另取同龄C5B7L/6J小鼠9只为正常对照组。各组灌胃60 d相应药物后,采用Morris水迷宫观察各组小鼠空间认知能力;逆转录-聚合酶链反应(qRT-PCR)法检测CK1、PP5和HSP90 mRNA表达情况;Western印迹法检测CK1、PP5和HSP90蛋白表达情况。结果与模型组相比,TSG能够提高小鼠空间认知能力;降低CK1、HSP90及提高PP5的mRNA表达水平(P<0.05,P<0.01);降低CK1和HSP90和提高PP5的蛋白表达水平(P<0.05,P<0.01)。结论TSG治疗阿尔茨海默病的机制可能与降低CK1和HSP90的表达水平与蛋白表达水平,提高PP5的表达水平与蛋白表达水平等机制有关。

PGK1-coupled HSP90 stabilizes GSK3βexpression to regulate the stemness of breast cancer stem cells

Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.

Hsp90α作为肿瘤标志物的应用前景

热休克蛋白（heat shock protein，HSP）是细胞或生物体在受到热胁迫等应激后，新合成或合成量增加的一类分子伴侣蛋白质。这类蛋白质在生物进化过程中高度保守，并广泛分布于原核细胞、真核细胞各物种中。它们具有多种生物活性，其中最基础的作用是保护蛋白质的正确空间结构、防止其意外降解，保持蛋白质正常功能。热休克蛋白90α（heat shock protein 90α， HSP90α）是热休克蛋白家族重要的成员之一。近年来，随着国内外对于HSP90α研究的不断进展，越来越多的实验证据表明，HSP90α与多种类型肿瘤的发生、发展以及肿瘤患者的预后等都有密切关系。

热休克蛋白27,60和90在胃癌中表达及其临床价值

目的:探讨HSP-27、-60和-90在胃癌中的表达及其临床意义。方法:采用免疫组织化学检测66例胃癌组织中HSP-27、-60和-90的表达情况,并结合临床病理特征、肿瘤增殖能力和患者生存期分析三种热休克蛋白表达的临床意义。结果:HSP-27、-60和-90在胃癌组织中异常高表达。HSP-27表达与肿瘤大小(p T,P=0.026)、器官转移(p M,P=0.046)及病理分期(P=0.041)相关,而HSP-27染色强度与淋巴腺状态明显相关(p N,P=0.042)。HSP-60表达与患者性别相关(P=0.011),而HSP-60染色强度与患者年龄(P=0.027)和肿瘤病理组织学分级(P=0.031)相关。HSP-90表达与本研究分析的临床病理参数无相关性;但是,HSP-90染色强度与肿瘤大小存在着显著关系(p T,P=0.020)。单因素分析表明HSP-90高表达与生存期更长显著相关(P=0.033),多因素分析证实HSP-90高表达是胃癌独立预后因素(P=0.026)。结论:HSP-27、-60和-90与某些临床病理参数有关,这些参数对于胃腺癌患者的治疗至关重要。胃腺癌患者HSP-90高表达是独立预后指标。

Hsp90βis critical for the infection of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.

Effects of heat shock on change of HSC70/HSP68,acid and alkaline phosphatases before and after rat partial hepatectomy

INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can be induced to proliferatequickly by partial hepatectomy (PH),and,to somedegree,they stop dividing and re-differentiate intocells functioning as hepatocytes.This shows

Hsp90 Is Involved in the Regulation of Cytosolic =recursor Protein Abundance in Tomato

Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 （Hsp70） and 90 （Hsp90） assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein （Chip）, appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.

Heat-shock protein 90 in Candida albicans

Researches on Candidal heat-shock protein 90 (HSP90) in recent years are summarized. Candida albicans is a commensal pathogen in human and animals. in immunocompromised individuals it behaves as an opportunist pathogen, giving rise to superficial or systemic infections. Systemic candidosis is a common cause of death among immunocompromised and debilitated patients, in which the mortality is as high as 70%. HSP90 is now recognized as an immunodominant antigen in C. albicans and plays a key role in systemic candidosis as a molecular chaperone. The 47-ku peptide is the breakdown product of HSP90. Patients who has recovered from systemic candidosis produce high titre of antibodies to 47-ku antigen, whereas the fatal cases have little antibody or falling titre? The three commonest epitopes of candidal HSP90 have been mapped, epitopes C, B and H. Epitopes C and H are immunogenic. The antibody probes of both epitopes may be developed into a new serological test agents for systemic candidosis due to rather

热休克蛋白70在神经元可塑性中作用的研究进展

热休克蛋白（heat shock protein，HSP）是体内一族重要的蛋白质，不仅具有帮助蛋白质正确组装、折叠、转运的作用，也参与抑制凋亡，并与抗原提呈、甾体激素受体功能、细胞内运输、核受体结合有关。热休克蛋白包括小分子HSP家族、HSP60家族、HSP70家族、HSP90家族和HSP110家族，其中HSP70家族包括HSC70、

热休克蛋白-肽复合物与肿瘤免疫

热休克蛋白（heat shock protein,HSP）是一类在生物进化过程中高度保守,广泛表达于所有生命机体组织细胞并具有管家功能的蛋白质。根据分子量的差异,HSP可分为10个家族,包括HSP110/grp170、gp96/grp94、HSP90、HSP70/grp78、HSP65、HSP25/27等,每个家族有1~5个成员[1]。在正常生理状态下,

硼替佐米对人类多发性骨髓瘤细胞系U266细胞抗凋亡蛋白HSP-90的影响

目的 探讨硼替佐米对人类多发性骨髓瘤（MM）细胞系U266细胞抗凋亡蛋白HSP-90的影响.方法 采用RT-PCR技术研究经不同浓度的硼替佐米作用于U266细胞4 h后其内HSP-90mRNA表达情况的变化.结果 随着硼替佐米浓度的增加,其MM细胞中HSP-90α的mRNA表达量逐渐增加.由低浓度组到高浓度组（0、50、100、150、200 nmol/L）所对应的HSP-90α的灰度值分别为0.343±0.017、0.505±0.039、0.640±0.029、0.760±0.059、0.963±0.054;两两组间比较差异有统计学意义（P〈0.05）.浓度由低到高组对应HSP-90β的灰度值分别为0.610±0.022、0.765±0.050、0.645±0.052、0.770±0.059、0.790±0.027,而HSP-90β的灰度值0 nmol/L组与50、150、200nmol/L组之间差异有统计学意义（P〈0.05）.50、100 nmol/L浓度组对应的HSP-90β的灰度值不同（P〈0.05）.100 nmol/L组与150、200 nmol/L组对应的HSP-90β的灰度值差异有统计学意义（P〈0.05）.HSP-90β的mRNA表达量增加趋势不很明显,但总体差异仍具统计学意义（P〈0.05）.结论 硼替佐米可以上调HSP-90的分子水平的表达,并且以上调HSp-90α的表达为主.

小儿肾母细胞瘤HSP70和HSP90α的表达及其意义

目的检测热休克蛋白70(heatshockprotein,HSP)和90α在小儿肾母细胞瘤组织中的表达,并探讨其临床意义。方法应用免疫组织化学方法及逆转录聚合酶链反应(RTPCR)技术检测20例肾母细胞瘤组织及瘤旁正常肾脏组织中HSP70和HSP90α的表达,其中男8例,女12例,年龄19个月至5岁,平均(22.5±13.7)个月。按NWTS3组织分型标准,20例肾母细胞瘤均为预后好的组织结构,包括间叶型5例,胚芽型9例,上皮型6例。临床分期Ⅰ期4例,Ⅱ期8例,Ⅲ期6例,Ⅳ期2例。结果免疫组化结果示HSP70和HSP90α在肿瘤组织中的阳性表达率(75.0%,15/20;80.0%,16/20)明显低于正常肾组织(100%,20/20;100%,20/20),P值分别为0.017和0.035,主要位于胚芽及上皮成分,而肿瘤基质均为阴性。对照组HSP70和HSP90α的表达位于肾小管上皮细胞、肾小球系膜细胞及内皮细胞胞浆内,均为强阳性。RTPCR结果示对照组HSP70和HSP90αmRNA的扩增产物条带多为阳性或强阳性,而在肾母细胞瘤中多为中度阳性或弱阳性表达。HSP70/βactin在对照组和肿瘤组分别为1.41±0.26和0.73±0.27(P=0.004),HSP90α/βactin在对照组和肿瘤组分别为0.95±0.04和0.61±0.13(P=0.0009)。相关分析表明HSP70和HSP90α蛋白和mRNA的表达均成正相关(r=0.694,P=0.001;r=0.720,P=0.001)。HSP70。

RNA干扰技术沉默热休克蛋白70对喉癌Hep-2细胞增殖的影响

喉癌是上呼吸道常见的恶性肿瘤,严重影响着人类的健康。研究表明热休克蛋白70(heat shock protein 70,HSP70)在正常组织中多不表达,但在包括头颈鳞状细胞癌在内的多种肿瘤组织却有异常表达。肿瘤细胞中高表达的HSP70,对细胞的增殖、侵袭及转移、机体对肿瘤治疗耐受性的发生及肿瘤预后等方面均有很重要的作用[1]。RNA干扰(RNA interference,RNAi)技术是近年来发展起来的一项新的基因技术。