As a critical apoptosis executioner, caspase-3 becomes activated and then enters into the nucleus to exert its function. However, the molecular mechanism of this nuclear entry of active caspase-3 is still unknown. In ...As a critical apoptosis executioner, caspase-3 becomes activated and then enters into the nucleus to exert its function. However, the molecular mechanism of this nuclear entry of active caspase-3 is still unknown. In this study, we revealed that easpase-3 harbors a crm-l-independent nuclear export signal （NES） in its small subunit. Using reversecaspase-3 as the study model, we found that the function of the NES in caspase-3 was not disturbed by the conformational changes during induced caspase-3 activation. Mutations disrupting the cleavage activity or p3-recognition site resulted in a defect in the nuclear entry of active caspase-3. We provide evidence that the p3-mediated specific cleavage activity of active caspase-3 abrogated the function of the NES. In conclusion, our results demonstrate that during caspase-3 activation, NES is constitutively present, p3-mediated specific cleavage activity abrogates the NES function in caspase-3, thus facilitating the nuclear entry of active caspase-3.展开更多
目的探讨血清Caspase切割的细胞角蛋白18(CCCK-18)表达水平对脑梗死患者重组组织型纤溶酶原激活剂(r-tPA)静脉溶栓后出血转化(HT)风险的预测价值。方法93例脑梗死r-tPA静脉溶栓患者,根据是否发生HT分为HT组(42例)及未HT组(51例)。利用...目的探讨血清Caspase切割的细胞角蛋白18(CCCK-18)表达水平对脑梗死患者重组组织型纤溶酶原激活剂(r-tPA)静脉溶栓后出血转化(HT)风险的预测价值。方法93例脑梗死r-tPA静脉溶栓患者,根据是否发生HT分为HT组(42例)及未HT组(51例)。利用酶联免疫吸附试验法(ELISA)检测血清CCCK-18水平,比较两组的年龄、性别、体质量指数、吸烟史、饮酒史、高脂血症史、糖尿病史、高血压史、甘油三酯(TG)、总胆固醇(TC)、血清CCCK-18水平及美国国立卫生研究院卒中量表(NIHSS)评分,并利用多因素Logistic回归分析脑梗死患者溶栓后发生HT的危险因素;利用受试者工作特征曲线(ROC)评价血清CCCK-18水平、NIHSS评分对溶栓后发生HT的诊断价值。结果两组患者年龄、性别、体质量指数、TG水平及吸烟史、饮酒史、高脂血症史、糖尿病史比例比较,差异无统计学意义(P>0.05);HT组患者TC水平低于未HT组、高血压史比例高于未HT组,差异有统计学意义(P<0.05)。治疗后3 d,HT组患者血清CCCK-18、NIHSS评分均高于治疗前,未HT组患者血清CCCK-18、NIHSS评分均明显低于治疗前,差异有统计学意义(P<0.05);且治疗后3 d HT组患者血清CCCK-18(241.76±28.46)U/L、NIHSS评分(14.73±2.69)分均高于未HT组的(156.73±26.59)U/L、(7.21±1.32)分,差异有统计学意义(P<0.05)。经多因素Logistic回归分析,结果显示有高血压史、治疗前高CCCK-18水平、治疗前高NIHSS评分是脑梗死患者静脉溶栓后发生HT的危险因素(P<0.05)。ROC结果显示,治疗前血清CCCK-18、NIHSS评分预测脑梗死r-tPA静脉溶栓后HT的曲线下面积(AUC)分别为0.820(95%CI:0.726,0.872)、0.824(95%CI:0.731,0.875),二者联合预测的AUC为0.902(95%CI:0.821,0.947)。结论脑梗死r-tPA静脉溶栓后HT患者血清CCCK-18水平呈高水平表达,对患者有一定的预测价值,可能成为指示脑梗死r-tPA静脉溶栓后发生HT的潜在指标,且二者联合预测的价值更高。展开更多
AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cel...AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.展开更多
AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures compri...AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.展开更多
基金Supplementary information is linked to the online version of the paper on the Cell Research website.Acknowledgments We thank Prof Jian Wang （Shanghai University, Shanghai） for his valuable revision and discussion. This work was supported by grants from the National Natural Science Foundation of China （30700411）, Shenzhen Bureau of Science Technology and Information （SZKJ-2006018, SZKJ-2007012）, Shenzhen Nanshan Bureau of Science Technology and Information （2008036） and Shenzhen Key Laboratory Advancement Scheme.
文摘As a critical apoptosis executioner, caspase-3 becomes activated and then enters into the nucleus to exert its function. However, the molecular mechanism of this nuclear entry of active caspase-3 is still unknown. In this study, we revealed that easpase-3 harbors a crm-l-independent nuclear export signal （NES） in its small subunit. Using reversecaspase-3 as the study model, we found that the function of the NES in caspase-3 was not disturbed by the conformational changes during induced caspase-3 activation. Mutations disrupting the cleavage activity or p3-recognition site resulted in a defect in the nuclear entry of active caspase-3. We provide evidence that the p3-mediated specific cleavage activity of active caspase-3 abrogated the function of the NES. In conclusion, our results demonstrate that during caspase-3 activation, NES is constitutively present, p3-mediated specific cleavage activity abrogates the NES function in caspase-3, thus facilitating the nuclear entry of active caspase-3.
文摘目的探讨血清Caspase切割的细胞角蛋白18(CCCK-18)表达水平对脑梗死患者重组组织型纤溶酶原激活剂(r-tPA)静脉溶栓后出血转化(HT)风险的预测价值。方法93例脑梗死r-tPA静脉溶栓患者,根据是否发生HT分为HT组(42例)及未HT组(51例)。利用酶联免疫吸附试验法(ELISA)检测血清CCCK-18水平,比较两组的年龄、性别、体质量指数、吸烟史、饮酒史、高脂血症史、糖尿病史、高血压史、甘油三酯(TG)、总胆固醇(TC)、血清CCCK-18水平及美国国立卫生研究院卒中量表(NIHSS)评分,并利用多因素Logistic回归分析脑梗死患者溶栓后发生HT的危险因素;利用受试者工作特征曲线(ROC)评价血清CCCK-18水平、NIHSS评分对溶栓后发生HT的诊断价值。结果两组患者年龄、性别、体质量指数、TG水平及吸烟史、饮酒史、高脂血症史、糖尿病史比例比较,差异无统计学意义(P>0.05);HT组患者TC水平低于未HT组、高血压史比例高于未HT组,差异有统计学意义(P<0.05)。治疗后3 d,HT组患者血清CCCK-18、NIHSS评分均高于治疗前,未HT组患者血清CCCK-18、NIHSS评分均明显低于治疗前,差异有统计学意义(P<0.05);且治疗后3 d HT组患者血清CCCK-18(241.76±28.46)U/L、NIHSS评分(14.73±2.69)分均高于未HT组的(156.73±26.59)U/L、(7.21±1.32)分,差异有统计学意义(P<0.05)。经多因素Logistic回归分析,结果显示有高血压史、治疗前高CCCK-18水平、治疗前高NIHSS评分是脑梗死患者静脉溶栓后发生HT的危险因素(P<0.05)。ROC结果显示,治疗前血清CCCK-18、NIHSS评分预测脑梗死r-tPA静脉溶栓后HT的曲线下面积(AUC)分别为0.820(95%CI:0.726,0.872)、0.824(95%CI:0.731,0.875),二者联合预测的AUC为0.902(95%CI:0.821,0.947)。结论脑梗死r-tPA静脉溶栓后HT患者血清CCCK-18水平呈高水平表达,对患者有一定的预测价值,可能成为指示脑梗死r-tPA静脉溶栓后发生HT的潜在指标,且二者联合预测的价值更高。
文摘AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.
基金Supported by NIH Grant DK068097funds provided by Rhode Island Hospital+1 种基金the Deutsche Forschungsgemeinschaft grant (DFG) grant GE1193/1-1NIH COBRE Award (RR-P20 RR17695)
文摘AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.
