The genes(gsh\|I,gsh\|II)for γ\|glutamyl\|cysteine synthetase(GSH\|I) and glutathione synthetase(GSH\|II)from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragme...The genes(gsh\|I,gsh\|II)for γ\|glutamyl\|cysteine synthetase(GSH\|I) and glutathione synthetase(GSH\|II)from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc\|gsh. E.coli BL21 was transformed by pTrc\|gsh for expression of the related enzymes.Analysis of SDS\|PAGE showed that the expected products were expressed. E.coli BL21(pTrc\|gsh) were incubated at 37℃ and pH 7.2 to OD 550 =0 5.The conditions were then switched to 34℃ and pH6.7 after the addition of 0.1mmol/L IPTG.The expressed products were up to 25% of the total protein of the bacteria.Acetone\|treated cells of the engineered strain could synthesize GSH efficiently.展开更多
Nrf2（NF-E2 related factor）在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用，是外源性有毒物质和氧化应激的感受器。细胞质Keap1蛋白是Nrf2的负调节因子。Nrf2能调控Ⅱ相解毒酶基因如γ谷氨酰半胱氨酸合...Nrf2（NF-E2 related factor）在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用，是外源性有毒物质和氧化应激的感受器。细胞质Keap1蛋白是Nrf2的负调节因子。Nrf2能调控Ⅱ相解毒酶基因如γ谷氨酰半胱氨酸合成酶（γ-GCS）的表达。氧化／抗氧化失衡是慢性阻塞性肺疾病的主要发病机制。谷胱甘肽（GSH）是肺内主要的抗氧化剂，而γ-GCS是GSH的限速酶，因此通过Nrf2途径调控细胞内γ-GCS的表达具有重要作用。展开更多
目的探讨慢性阻塞性肺疾病(简称慢阻肺)患者肺组织中活化转录因子3(ATF3)、活化转录因子4(ATF4)的表达变化,以及ATF3和ATF4对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨其在慢阻肺发病中的作用。方法收集伴或不伴慢阻肺并行肺叶...目的探讨慢性阻塞性肺疾病(简称慢阻肺)患者肺组织中活化转录因子3(ATF3)、活化转录因子4(ATF4)的表达变化,以及ATF3和ATF4对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨其在慢阻肺发病中的作用。方法收集伴或不伴慢阻肺并行肺叶切除的肺癌患者临床肺组织标本40例(慢阻肺组和对照组各20例),取材标本均为远离原发肺癌病灶5 cm以上、肉眼观察无肺癌浸润的外周肺组织。所有患者术前均详细询问病史,进行体格检查、胸部X线片或肺部CT以及肺功能检测。应用原位杂交及免疫组化检测患者肺组织中ATF3、ATF4、γ-GCS重链亚基(γ-GCS-HS)m RNA及蛋白的表达,应用免疫共沉淀法(CO-IP)检测ATF3、ATF4与γ-GCS-HS蛋白之间的相互作用,并对ATF3、ATF4 m RNA及蛋白与γ-GCS-HS m RNA及蛋白进行相关分析。结果慢阻肺患者肺组织中ATF3、ATF4、γ-GCS-HS m RNA及蛋白呈强阳性表达,较对照组显著升高(P<0.01)。在γ-GCS-HS抗体捕获的免疫沉淀中,ATF3、ATF4抗体均可杂交出明显的蛋白条带,且慢阻肺组较对照组明显增强(P<0.01)。相关分析显示肺组织中ATF3、ATF4蛋白表达与γ-GCS-HS m RNA及蛋白表达均呈明显正相关(P<0.01);ATF3、ATF4、γ-GCS-HS蛋白表达与FEV1%pred、FEV1/FVC均呈明显正相关(P<0.01)。结论在慢阻肺患者发病过程中,氧化应激诱导转录因子ATF3和ATF4过表达,ATF3和ATF4可能通过影响γ-GCS m RNA和蛋白的表达而发挥抗氧化保护作用。展开更多
The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1(NQO1)and y-glutamylcysteine synthetase(γ-GCS)in brain tissue...The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1(NQO1)and y-glutamylcysteine synthetase(γ-GCS)in brain tissues after intracerebral hemorrhage(ICH).The microglial cells obtained from newborn rats were cultured and then randomly divided into the normal control group(NC group),model control group(MC group),rosiglitazone(RSG)intervention group(RSG group),retinoic-acid intervention group(RSG+RA group),and sulfbraphane group(RSG+SF group).The expression levels of NQO1,γ-GCS,and nuclear factor E2-related factor 2(Nrf2)were measured by real-time polymerase chain reaction(RT-PCR)and Western blotting,respectively.The results showed that the levels of NQO1,γ-GCS and Nrf2 were significantly increased in the MC group and the RSG group as compared with those in the NC group(P<0.01).They were found to be markedly decreased in the RSG+RA group and increased in the RSG+SF group when compared with those in the MC group or the RSG group(P<0.01).The RSG+SF group displayed the highest levels of NQO1,γ-GCS,and Nrf2 among the five groups.In conclusion,a medium dose of RSG increased the anti-oxidative ability of thrombinactivated microglia by increasing the expression of NQO1 and γ-GCS.The molecular mechanisms underlying the increase of NQO1 and γ-GCS in thrombin-activated microglia may be associated with the activation of Nrf2.展开更多
文摘The genes(gsh\|I,gsh\|II)for γ\|glutamyl\|cysteine synthetase(GSH\|I) and glutathione synthetase(GSH\|II)from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc\|gsh. E.coli BL21 was transformed by pTrc\|gsh for expression of the related enzymes.Analysis of SDS\|PAGE showed that the expected products were expressed. E.coli BL21(pTrc\|gsh) were incubated at 37℃ and pH 7.2 to OD 550 =0 5.The conditions were then switched to 34℃ and pH6.7 after the addition of 0.1mmol/L IPTG.The expressed products were up to 25% of the total protein of the bacteria.Acetone\|treated cells of the engineered strain could synthesize GSH efficiently.
文摘Nrf2（NF-E2 related factor）在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用，是外源性有毒物质和氧化应激的感受器。细胞质Keap1蛋白是Nrf2的负调节因子。Nrf2能调控Ⅱ相解毒酶基因如γ谷氨酰半胱氨酸合成酶（γ-GCS）的表达。氧化／抗氧化失衡是慢性阻塞性肺疾病的主要发病机制。谷胱甘肽（GSH）是肺内主要的抗氧化剂，而γ-GCS是GSH的限速酶，因此通过Nrf2途径调控细胞内γ-GCS的表达具有重要作用。
文摘目的探讨慢性阻塞性肺疾病(简称慢阻肺)患者肺组织中活化转录因子3(ATF3)、活化转录因子4(ATF4)的表达变化,以及ATF3和ATF4对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨其在慢阻肺发病中的作用。方法收集伴或不伴慢阻肺并行肺叶切除的肺癌患者临床肺组织标本40例(慢阻肺组和对照组各20例),取材标本均为远离原发肺癌病灶5 cm以上、肉眼观察无肺癌浸润的外周肺组织。所有患者术前均详细询问病史,进行体格检查、胸部X线片或肺部CT以及肺功能检测。应用原位杂交及免疫组化检测患者肺组织中ATF3、ATF4、γ-GCS重链亚基(γ-GCS-HS)m RNA及蛋白的表达,应用免疫共沉淀法(CO-IP)检测ATF3、ATF4与γ-GCS-HS蛋白之间的相互作用,并对ATF3、ATF4 m RNA及蛋白与γ-GCS-HS m RNA及蛋白进行相关分析。结果慢阻肺患者肺组织中ATF3、ATF4、γ-GCS-HS m RNA及蛋白呈强阳性表达,较对照组显著升高(P<0.01)。在γ-GCS-HS抗体捕获的免疫沉淀中,ATF3、ATF4抗体均可杂交出明显的蛋白条带,且慢阻肺组较对照组明显增强(P<0.01)。相关分析显示肺组织中ATF3、ATF4蛋白表达与γ-GCS-HS m RNA及蛋白表达均呈明显正相关(P<0.01);ATF3、ATF4、γ-GCS-HS蛋白表达与FEV1%pred、FEV1/FVC均呈明显正相关(P<0.01)。结论在慢阻肺患者发病过程中,氧化应激诱导转录因子ATF3和ATF4过表达,ATF3和ATF4可能通过影响γ-GCS m RNA和蛋白的表达而发挥抗氧化保护作用。
基金grants from the National Natural Science Foundation of China(No.81560222)the Guizhou Science and Technology Foundation(No.[2017]7187,and No.[2013]2043).
文摘The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1(NQO1)and y-glutamylcysteine synthetase(γ-GCS)in brain tissues after intracerebral hemorrhage(ICH).The microglial cells obtained from newborn rats were cultured and then randomly divided into the normal control group(NC group),model control group(MC group),rosiglitazone(RSG)intervention group(RSG group),retinoic-acid intervention group(RSG+RA group),and sulfbraphane group(RSG+SF group).The expression levels of NQO1,γ-GCS,and nuclear factor E2-related factor 2(Nrf2)were measured by real-time polymerase chain reaction(RT-PCR)and Western blotting,respectively.The results showed that the levels of NQO1,γ-GCS and Nrf2 were significantly increased in the MC group and the RSG group as compared with those in the NC group(P<0.01).They were found to be markedly decreased in the RSG+RA group and increased in the RSG+SF group when compared with those in the MC group or the RSG group(P<0.01).The RSG+SF group displayed the highest levels of NQO1,γ-GCS,and Nrf2 among the five groups.In conclusion,a medium dose of RSG increased the anti-oxidative ability of thrombinactivated microglia by increasing the expression of NQO1 and γ-GCS.The molecular mechanisms underlying the increase of NQO1 and γ-GCS in thrombin-activated microglia may be associated with the activation of Nrf2.
