Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.     

Safety profile of the adjuvanted recombinant zoster vaccine: Pooled analysis of two large randomised phase 3 trials

Background

The ZOE-50 (NCT01165177) and ZOE-70 (NCT01165229) phase 3 clinical trials showed that the adjuvanted recombinant zoster vaccine (RZV) was ≥90% efficacious in preventing herpes zoster in adults. Here we present a comprehensive overview of the safety data from these studies.

Immunohistochemical and molecular analysis of PI3K/AKT/mTOR pathway in esophageal carcinoma

Among the numerous signaling pathways involved in tumorigenesis, PI3K‐AKT‐mTOR is a key one that regulates diverse cellular functions. However, its prognostic value in esophageal carcinoma remains unclear. In our study, we examined the immunohistochemical expression of phosphorylated (p‐) AKT, mTOR, p70S6K and 4E‐BP1 along with the mutational status of PIK3CA and AKT1 genes by High Resolution Melting Analysis and Pyrosequencing in 44 esophageal carcinomas. The results were correlated with the clinicopathological characteristics of the patients in an effort to define their possible prognostic significance. Total p‐mTOR cytoplasmic expression, assessed in 10 random areas, was positively correlated with tumor stage (Kruskal–Wallis ANOVA, I/II vs III/IV, p = 0.0500). Μoreover, maximum p‐mTOR cytoplasmic immunoexpression, estimated in hot spot areas, was positively associated with tumor grade (Mann–Whitney U test, I/II vs III, p = 0.0565). Interestingly, p‐4E‐BP1 immunoreactivity was negatively correlated with tumor histological grade (Mann–Whitney U test, I/II vs III, p = 0.0427). No mutation was observed in exons 9 and 20 of PIK3CA gene and in exon 4 of AKT1 gene. In conclusion, our findings depict the presence of activated PI3K/AKT/mTOR pathway in esophageal cancer bringing forward p‐mTOR and p‐4E‐BP1 for their potential role in esophageal carcinogenesis. Additional studies are warranted to validate our findings.     

局部脑缺血预处理对大鼠脑梗死的影响和HSP70的表达及mRNA的变化

目的建立局灶性可重复性大鼠脑缺血动物模型,研究短暂性局部脑缺血后再灌注不同时间对大脑中动脉阻塞(MCAO)的影响。方法应用改进的Longa's法,建立阻断左侧中动脉的局部脑缺血预处理模型。各组大鼠均经两次处理预处理(PC)组大鼠20min短暂脑缺血,分别在再灌注12h、1d、3d、5d、7d、14d后,造成MCAO;脑梗死组大鼠只在第二次处理造成大脑中动脉闭塞;短暂缺血组只在第一次处理时缺血20min,各组大鼠均在第二次处理后24h断头处死,检测以下指标神经功能缺失评分;TTC染色测量梗死范围HE染色观察组织结构变化;免疫印迹(原位杂交)和免疫组织化学染色观察HSP70的表达。结果PC后1d、2d、3d、5d、7d组与脑梗死组相比较,脑梗死范围,梗死周边区脑组织的缺血性损伤明显减轻了;短暂性脑缺血引起了轻微的神经细胞结构的改变并使缺血区HSP70的表达增加,MCAO后24hHSP70蛋白在缺血周边区出现了广泛表达。结论短暂局部缺血预处理可以诱导脑梗死后脑组织产生缺血耐受性,其保护作用出现再灌注后l~7d;缺血预处理引起HSP70的变化与缺血耐受的产生有一定联系。     

热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

目的探讨内源性热休克蛋白90（HSP90）在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶（AKT）相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型，将细胞分为正常组、缺氧组、加入HSP90特异性阻断剂格尔德霉素后再缺氧组（格尔德霉素＋缺氧组）。于缺氧后1、3、6、12、24、48h用噻唑蓝法检测心肌细胞的活力；缺氧24h，原位缺口末端标记法检测心肌细胞凋亡指数（AI）；缺氧1、3、6、12、24h，蛋白质印迹法检测大鼠心肌细胞中内源性HSP90及AKT表达水平。结果（1）缺氧24、48h，缺氧组、格尔德霉素＋缺氧组细胞活力均较正常组明显下降（P〈0．05）；格尔德霉素＋缺氧组细胞活力缺氧12h即开始明显下降，缺氧48h时明显低于缺氧组（P〈0．05）。（2）缺氧24h，缺氧组细胞AI为（10．7±1．2）％，明显高于正常组[（1．9±0．3）％．P〈0．05]；格尔德霉素＋缺氧组细胞AI为（26、3±5．3）％，明显高于缺氧组（P〈0．01）。（3）缺氧12h，缺氧组心肌细胞内源性HSP90及AKT表达水平高于正常组与格尔德霉素＋缺氧组；缺氧24h，缺氧组有所下降．格尔德霉素＋缺氧组则下降更明显。结论内源性HSP90对维持心肌细胞的活力有重要作用．缺氧心肌细胞AKT表达水平可受内源性HSP90表达水平的影响。     

Immunohistochemical expression of C-myc oncogene,heat shock protein 70 and HLA-DR molecules in malignant cutaneous melanoma

The clinical course of malignant melanomas is frequently unpredictable, although a number of prognostically useful variables can be identified. There is a need for additional markers of prognostic value. In a series of 60 malignant cutaneous melanomas, we analysed the immunohistochemical expression of c-myc proto-oncogene, heat shock protein 70 (HSP70) and HLA-DR molecules in order to investigate their prognostic significance. C-myc, HSP70 and HLA-DR were expressed in 43.3%, 56.6% and 38.3% of all melanoma cases, respectively. Advanced Clark levels (Clark III–V) were significantly associated with c-myc expression rate (P<0.05), HSP70 detection (P<0.01) and HLA-DR positivity (P<0.01). Increased Breslow thickness (>1.5 mm) was related to HLA-DR expression (P<0.05). High mitotic rate was closely associated with c-myc positivity (P<0.05), while HSP70 and HLA-DR expression separately correlated to clinical stage of the disease (P<0.05). The evaluation of these variables may be of immunological and prognostic significance. They were found to be associated with melanocyte subpopulations of the vertical growth phase which are arguably characterized by an increased invasive potential.     

Induction of HSP72 in Rat Liver by Chronic Ethanol Consumption Combined with Exercise: Association with the Prevention of Ethanol-Induced Fatty Liver by Exercise

Ethanol-induced fatty liver in rats was attenuated by repeated running exercise, and the protective effect of exercise was associated with the synergistic expression of heat shock proteins (HSP72). Rats were placed in four groups of six. The two ethanol-fed groups of rats received a liquid diet (Lieber-DeCarli formulation) in which 36% of the calories were derived from ethanol. One group remained sedentary (S/E), whereas the other was trained to run on a rodent treadmill at a speed of 27 m/min, 1 hr/day, 5 days/week, for 7 weeks (R/E). Two other groups–one exercised as previously mentioned (R/C) and one sedentary (S/C)–received control-liquid diets in which the ethanol was isocalorically substituted with a dextran/maltose mixture. The degree of fatty infiltration in liver sections stained with hematoxylin and eosin was graded on a 0–4 scale and the data analyzed by ANOVA on ranks. Ethanol significantly induced fatty infiltration in the S/E group, whereas fatty infiltration in the livers of the R/E group was not different from the S/C group. Electrophoresis and Western blotting of liver homogenates demonstrated that HSP72 was not expressed in either the S/C or S/E groups and was only slightly expressed in the R/C group. The combination of exercise and ethanol, however, resulted in an elevated expression of HSP72 in the R/E group. The content of HSP73 was unaffected by any treatment.