作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基...作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基供体尚不明确。本文通过比较超表达TMEM68对不同脂酰链饱和度的甘油酯、脂肪酸和甘油磷脂的作用,发现超表达TMEM68对不同饱和度的三酰甘油、二酰甘油、脂肪酸、磷脂酰胆碱和磷脂酰乙醇胺及其醚脂表现出不同的影响,并且这些脂质的变化存在一定的相关性;通过DGAT抑制剂处理,发现TMEM68不依赖DGAT活性合成三酰甘油,促进脂滴形成;通过分子对接分析,发现TMEM68与磷脂:二酰甘油酰基转移酶具有相似甚至更强的针对磷脂酰胆碱和磷脂酰乙醇胺及其醚脂的结合力。这些结果提示,TMEM68以二酰甘油为酰基受体,可能利用甘油磷脂作为酰基供体合成三酰甘油。展开更多
Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylgly...Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylglycerol acyltransferase(DGAT).Methods Effects of exogenous fatty acid and monoacylglycerol on TAG synthesis and lipid droplet(LD)formation in TMEM68 overexpression and knockout cells treated with DGAT inhibitor or not were investigated by comparing LD morphology,Oil Red O staining,and measurement of TAG levels.LDs were stained with fluorescence dye and observed by confocal fluorescence microscopy.TAG levels were determined with an enzyme-based triglyceride assay kit.Colocalization of TMEM68 and DGAT1 was detected by co-expression and confocal fluorescence microscopy and their interaction was determined by co-immunoprecipitation.RT-qPCR and immunoblotting assay were used to detect the expression of DGAT1.Results The synthesis of TAG catalyzed by TMEM68 was independent of DGAT activity.Surplus exogenous fatty acids and monoacylglycerol promoted TAG synthesis mainly through DGAT in human neuroblastoma cells.The LDs formed by TMEM68 were different in morphology from those by DGAT.In addition,TMEM68 and DGAT1 colocalized in the same endoplasmic reticulum(ER)compartment but did not interact physically.TMEM68 overexpression reduced the expression of DGAT1,the major DGAT enzyme involved in TAG synthesis,while TMEM68 knockout had little impact.Conclusion The TMEM68-mediated TAG synthesis pathway has distinct features from the canonical DGAT pathway,however,TMEM68 and DGAT may coregulate intracellular TAG levels.展开更多
野大豆(Glycine soja Siebold&Zucc.)是一种重要的粮油兼用作物,富含多种黄酮和黄酮糖苷,具有多种药理活性。糖基化可以将黄酮苷元转化为更稳定、生物活性更强、结构更多样的糖苷。通过基因组分析和酶挖掘,报道了一种来自野大豆的黄...野大豆(Glycine soja Siebold&Zucc.)是一种重要的粮油兼用作物,富含多种黄酮和黄酮糖苷,具有多种药理活性。糖基化可以将黄酮苷元转化为更稳定、生物活性更强、结构更多样的糖苷。通过基因组分析和酶挖掘,报道了一种来自野大豆的黄酮UDP-糖基转移酶GsUGT1,该酶主要作用于甘草素7-OH位置,对6种黄酮受体具有催化活性。此外,在大肠杆菌中成功异源表达GsUGT1并对其纯化,酶学性质表征发现在pH为7.0、温度为35℃时,酶的催化活性达到最大,米氏动力学研究表明GsUGT1的催化效率k_(cat)/K_(m)为6.70×10^(-5)μmol/(L·s)。最后通过分子对接对GsUGT1的催化机制进行了初步探究。首次报道了野大豆来源的UDP-糖基转移酶GsUGT1,该酶有望成为酶法合成黄酮糖苷衍生物的有效工具。展开更多
Glutathione S-transferases(GSTs) are a family of soluble detoxification enzymes which use reduced glutathione(GSH) in conjugation and reduction reactions.Toxic electrophiles,including a variety of carcinogens,are subs...Glutathione S-transferases(GSTs) are a family of soluble detoxification enzymes which use reduced glutathione(GSH) in conjugation and reduction reactions.Toxic electrophiles,including a variety of carcinogens,are substrates for the GSTs and are more easily excreted into bile or urine after conjugation or reduction .The three-dimensional structures of GSTs from several species,including humans,have been determined.GST activity has been found to present in all human tissues,and expression of the various GST isoenzymes differs in degree in the various tissues. Glutathione S-transferases may play a role in the protection of cells against toxic electrophiles and in the resistance to cancer chemotherapeutic agants.The polymorphisms of GSTs genes are one of the important factors that exercise influence on individual susceptibility to cancer.展开更多
目的分别对自建生化检测系统(简称自建系统)和配套生化检测系统(简称配套系统)进行初步性能评价。方法依照美国临床实验室标准化委员会(NCCLS)EP10-A2文件,使用自建系统(OLYMPUS AU400生化分析仪、罗氏cfas校准品及国产试剂)和配套系统(...目的分别对自建生化检测系统(简称自建系统)和配套生化检测系统(简称配套系统)进行初步性能评价。方法依照美国临床实验室标准化委员会(NCCLS)EP10-A2文件,使用自建系统(OLYMPUS AU400生化分析仪、罗氏cfas校准品及国产试剂)和配套系统(OLYMPUS AU 400生化分析仪、OLYMPUS校准品及OLYMPUS原装试剂)测定天门冬氨酸氨基转移酶(AST)、葡萄糖(Glu)、γ-谷氨酰基转移酶(GGT)、丙氨酸氨基转移酶(ALT)4个项目,计算其偏差、总不精密度及其截距、斜率、非线性、互染率、漂移性,并与各项目对应设定值作比较。结果自建系统:ALT的低值偏差和Glu的中、高值偏差超出允许范围,ALT的低值总不精密度超出允许范围,其余项目的偏差和总不精密度均在允许范围之内。GGT、ALT的非线性、截距和斜率以及Glu的斜率差异有统计学意义,其余项目的截距、斜率、非线性、互染率、漂移性差异均无统计学意义。配套系统:ALT的低值总不精密度超出允许范围,其余项目的偏差和总不精密度均在允许范围之内。GGT的截距和斜率差异有统计学意义,其余项目的截距、斜率、非线性、互染率、漂移性差异均无统计学意义。结论无论是使用自建系统测定AST、GGT、ALT,还是使用配套系统检测AST、Glu、GGT、ALT,偏差都在允许范围内,总不精密度小于美国临床实验室改进法案修正案(CLIA′88)能力验证计划中分析质量要求规定的总允许误差的1/3,互染率较低,稳定性较好。自建系统检测Glu的准确度有待加强改善。展开更多
文摘作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基供体尚不明确。本文通过比较超表达TMEM68对不同脂酰链饱和度的甘油酯、脂肪酸和甘油磷脂的作用,发现超表达TMEM68对不同饱和度的三酰甘油、二酰甘油、脂肪酸、磷脂酰胆碱和磷脂酰乙醇胺及其醚脂表现出不同的影响,并且这些脂质的变化存在一定的相关性;通过DGAT抑制剂处理,发现TMEM68不依赖DGAT活性合成三酰甘油,促进脂滴形成;通过分子对接分析,发现TMEM68与磷脂:二酰甘油酰基转移酶具有相似甚至更强的针对磷脂酰胆碱和磷脂酰乙醇胺及其醚脂的结合力。这些结果提示,TMEM68以二酰甘油为酰基受体,可能利用甘油磷脂作为酰基供体合成三酰甘油。
文摘Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylglycerol acyltransferase(DGAT).Methods Effects of exogenous fatty acid and monoacylglycerol on TAG synthesis and lipid droplet(LD)formation in TMEM68 overexpression and knockout cells treated with DGAT inhibitor or not were investigated by comparing LD morphology,Oil Red O staining,and measurement of TAG levels.LDs were stained with fluorescence dye and observed by confocal fluorescence microscopy.TAG levels were determined with an enzyme-based triglyceride assay kit.Colocalization of TMEM68 and DGAT1 was detected by co-expression and confocal fluorescence microscopy and their interaction was determined by co-immunoprecipitation.RT-qPCR and immunoblotting assay were used to detect the expression of DGAT1.Results The synthesis of TAG catalyzed by TMEM68 was independent of DGAT activity.Surplus exogenous fatty acids and monoacylglycerol promoted TAG synthesis mainly through DGAT in human neuroblastoma cells.The LDs formed by TMEM68 were different in morphology from those by DGAT.In addition,TMEM68 and DGAT1 colocalized in the same endoplasmic reticulum(ER)compartment but did not interact physically.TMEM68 overexpression reduced the expression of DGAT1,the major DGAT enzyme involved in TAG synthesis,while TMEM68 knockout had little impact.Conclusion The TMEM68-mediated TAG synthesis pathway has distinct features from the canonical DGAT pathway,however,TMEM68 and DGAT may coregulate intracellular TAG levels.
文摘野大豆(Glycine soja Siebold&Zucc.)是一种重要的粮油兼用作物,富含多种黄酮和黄酮糖苷,具有多种药理活性。糖基化可以将黄酮苷元转化为更稳定、生物活性更强、结构更多样的糖苷。通过基因组分析和酶挖掘,报道了一种来自野大豆的黄酮UDP-糖基转移酶GsUGT1,该酶主要作用于甘草素7-OH位置,对6种黄酮受体具有催化活性。此外,在大肠杆菌中成功异源表达GsUGT1并对其纯化,酶学性质表征发现在pH为7.0、温度为35℃时,酶的催化活性达到最大,米氏动力学研究表明GsUGT1的催化效率k_(cat)/K_(m)为6.70×10^(-5)μmol/(L·s)。最后通过分子对接对GsUGT1的催化机制进行了初步探究。首次报道了野大豆来源的UDP-糖基转移酶GsUGT1,该酶有望成为酶法合成黄酮糖苷衍生物的有效工具。
文摘Glutathione S-transferases(GSTs) are a family of soluble detoxification enzymes which use reduced glutathione(GSH) in conjugation and reduction reactions.Toxic electrophiles,including a variety of carcinogens,are substrates for the GSTs and are more easily excreted into bile or urine after conjugation or reduction .The three-dimensional structures of GSTs from several species,including humans,have been determined.GST activity has been found to present in all human tissues,and expression of the various GST isoenzymes differs in degree in the various tissues. Glutathione S-transferases may play a role in the protection of cells against toxic electrophiles and in the resistance to cancer chemotherapeutic agants.The polymorphisms of GSTs genes are one of the important factors that exercise influence on individual susceptibility to cancer.
文摘目的分别对自建生化检测系统(简称自建系统)和配套生化检测系统(简称配套系统)进行初步性能评价。方法依照美国临床实验室标准化委员会(NCCLS)EP10-A2文件,使用自建系统(OLYMPUS AU400生化分析仪、罗氏cfas校准品及国产试剂)和配套系统(OLYMPUS AU 400生化分析仪、OLYMPUS校准品及OLYMPUS原装试剂)测定天门冬氨酸氨基转移酶(AST)、葡萄糖(Glu)、γ-谷氨酰基转移酶(GGT)、丙氨酸氨基转移酶(ALT)4个项目,计算其偏差、总不精密度及其截距、斜率、非线性、互染率、漂移性,并与各项目对应设定值作比较。结果自建系统:ALT的低值偏差和Glu的中、高值偏差超出允许范围,ALT的低值总不精密度超出允许范围,其余项目的偏差和总不精密度均在允许范围之内。GGT、ALT的非线性、截距和斜率以及Glu的斜率差异有统计学意义,其余项目的截距、斜率、非线性、互染率、漂移性差异均无统计学意义。配套系统:ALT的低值总不精密度超出允许范围,其余项目的偏差和总不精密度均在允许范围之内。GGT的截距和斜率差异有统计学意义,其余项目的截距、斜率、非线性、互染率、漂移性差异均无统计学意义。结论无论是使用自建系统测定AST、GGT、ALT,还是使用配套系统检测AST、Glu、GGT、ALT,偏差都在允许范围内,总不精密度小于美国临床实验室改进法案修正案(CLIA′88)能力验证计划中分析质量要求规定的总允许误差的1/3,互染率较低,稳定性较好。自建系统检测Glu的准确度有待加强改善。
