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
猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方...猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。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.
文摘猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。
