近年来,人类免疫缺陷病毒(HIV)的检测工作在我国发展很快,各地已先后建立了上百个检测实验室,HIV检测的管理和质控也在逐渐加强.自1985年以来,检测HIV的初筛试剂不断发展.第三代试剂的出现大大缩短了从感染到检出抗体的时间[1].最近研...近年来,人类免疫缺陷病毒(HIV)的检测工作在我国发展很快,各地已先后建立了上百个检测实验室,HIV检测的管理和质控也在逐渐加强.自1985年以来,检测HIV的初筛试剂不断发展.第三代试剂的出现大大缩短了从感染到检出抗体的时间[1].最近研制出了第四代试剂,它能同时检测抗原和抗体.法国某公司生产的同时检测人类免疫缺陷病毒抗体和抗原的荧光酶联免疫(VIDAS HIV DUO)试剂,是一种能同时检测HIV 1+2型抗体和HIV-1 p24抗原的第四代试剂,用全自动荧光酶联免疫分析(VIDAS)仪进行检测.资料显示,VIDAS HIV DUO试剂比第三代试剂缩短了检出的时间,窗口期缩短为两个星期左右[2].2000年我们用全自动酶联免疫分析系统对部分血清进行了检测,结果如下.展开更多
AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which...AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this study.Human H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH expression.MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis.The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined.Next,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate.All of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged MUTYH.The mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of 8OHG.The mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2,1.91 × 10-2,and 1.96 × 10-2,respectively,meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.CONCLUSION:The suppressive activities of p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.展开更多
文摘近年来,人类免疫缺陷病毒(HIV)的检测工作在我国发展很快,各地已先后建立了上百个检测实验室,HIV检测的管理和质控也在逐渐加强.自1985年以来,检测HIV的初筛试剂不断发展.第三代试剂的出现大大缩短了从感染到检出抗体的时间[1].最近研制出了第四代试剂,它能同时检测抗原和抗体.法国某公司生产的同时检测人类免疫缺陷病毒抗体和抗原的荧光酶联免疫(VIDAS HIV DUO)试剂,是一种能同时检测HIV 1+2型抗体和HIV-1 p24抗原的第四代试剂,用全自动荧光酶联免疫分析(VIDAS)仪进行检测.资料显示,VIDAS HIV DUO试剂比第三代试剂缩短了检出的时间,窗口期缩短为两个星期左右[2].2000年我们用全自动酶联免疫分析系统对部分血清进行了检测,结果如下.
基金Supported by Grants from the Ministry of Health,Labour and Welfare(21-1)the Japan Society for the Promotion of Science (22590356 and 22790378)+3 种基金the Hamamatsu Foundation for Science and Technology Promotion,the Ministry of Education, Culture,Sports,Science and Technology(221S0001)the Takeda Science Foundationthe Aichi Cancer Research Foundationthe Smoking Research Foundation
文摘AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this study.Human H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH expression.MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis.The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined.Next,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate.All of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged MUTYH.The mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of 8OHG.The mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2,1.91 × 10-2,and 1.96 × 10-2,respectively,meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.CONCLUSION:The suppressive activities of p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.
