The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity bein...The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.展开更多
仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法...仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。展开更多
目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,...目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。展开更多
文摘The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.
文摘仙人掌X病毒(cactuSviruSX,CVX)严重威胁世界火龙果产业的发展。为实现对该病毒的定量检测,本研究根据CVX外壳蛋白(CP)的保守序列设计特异性引物CVX-F/-R,建立了引物浓度150 nmol/L,退火温度62℃的SYBR Green I实时荧光定量PCR检测方法。该引物扩增效率为97.38%,R^(2)为0.9965,对标准品的检测极限浓度为8×10^(2)拷贝/μL,其灵敏度是普通PCR的100倍。对火龙果不同组织中CVX的相对表达量进行检测发现,病毒在花丝中的含量最高,之后依次为花萼、花瓣、嫩枝、老枝和根。对采自贵州黔南州的40份火龙果样品进行检测,共检测出32份阳性样品。上述结果表明,本研究建立的实时荧光定量PCR特异性强、灵敏度高,为今后CVX的检测提供了重要的技术补充。
文摘目的建立炭疽芽孢杆菌的微滴式数字聚合酶链式反应(Droplet digital PCR,ddPCR)方法对炭疽芽孢杆菌实验室活动污染的定量评估提供技术支持。方法以炭疽芽孢杆菌pXO1质粒编码保护性抗原pagA基因为靶序列,优化微滴数字PCR方法的反应条件,建立实验室微环境中炭疽芽孢杆菌核酸定量方法;对比微滴式数字PCR方法和平板计数法的定量评估效果,分析ddPCR的灵敏性、特异性和重复性。结果建立的ddPCR方法最佳引物和探针终浓度分别为900nmol·L^(-1)和250nmol·L^(-1),最佳退火温度为60℃,最佳升降温速度为1℃/s,本方法的最低检测下限为1.12copies·μL^(-1),未发现与常见疫病存在交叉反应,重复性试验的变异系数小于5%。结论本研究中建立的炭疽芽孢杆菌的微滴数字PCR方法敏感性高、特异性强、重复性好,为疫情监测、流行病学调查和实验室污染微环境检测提供重要技术。
