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
文摘以菊糖芽孢乳杆菌(Sporolactobacillus inulinus)为出发菌株,经紫外线、硫酸二乙酯的诱变处理,采用碳酸钙平板初筛及摇瓶复筛得到1株高产D-乳酸的正向突变株D11。通过单因素试验研究D11菌株发酵产D-乳酸的条件。结果表明,当发酵温度37℃,初始p H 6.5,转速160 r/min,接种量5%,装液量50m L/250 m L,发酵68 h,该菌株D-乳酸产量达到56.18 g/L,比出发菌种提高49.53%。
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.
