[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase en...[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR.To evaluate the effect of ERG2 overexpression on the sterol content in the yeast,the expression plasmid pPERG2 was constructed and transformed into S.cerevisiae strain YS58 to generate recombinant strain YS58(pPERG2).In addition,the regulation role of sterol C-24 methyltransferase,encoded by ERG6,in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6,ERG2 respectively,or overexpressing ERG6 and ERG2 simultaneously.[Results] Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S.cerevisiae.Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis,overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level,which was 1.41-fold of that in empty vector strain,1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain.[Conclusion] These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S.cerevisiae.展开更多
文摘[Objective] Ergosterol is a fungal metabolite with economic importance.For ergosterol biosynthesis,to identify the bottleneck enzymes in the metabolic pathway is of crucial importance.[Methods] Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR.To evaluate the effect of ERG2 overexpression on the sterol content in the yeast,the expression plasmid pPERG2 was constructed and transformed into S.cerevisiae strain YS58 to generate recombinant strain YS58(pPERG2).In addition,the regulation role of sterol C-24 methyltransferase,encoded by ERG6,in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6,ERG2 respectively,or overexpressing ERG6 and ERG2 simultaneously.[Results] Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S.cerevisiae.Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis,overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level,which was 1.41-fold of that in empty vector strain,1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain.[Conclusion] These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S.cerevisiae.
文摘目的探讨新生儿持续肺动脉高压(persistent pulmonary hypertension of newborn,PPHN)患儿血清人CXC型趋化因子配体8(C-X-C motif chemokine ligand 8,CXCL8)、CXCL12与一氧化氮吸入治疗临床转归的关系。方法选择2021-08/2023-05月作者医院收治并给予一氧化氮吸入治疗的PPHN患儿135例为研究对象。根据患儿出院时临床转归结局分为死亡组(n=32)和存活组(n=103)。比较两组PPHN患儿血清CXCL8、CXCL12水平。单因素及多因素Logistic回归模型分析接受一氧化氮吸入治疗PPHN患儿临床转归的影响因素。受试者工作特征(receiver operating characteristic,ROC)曲线分析血清CXCL8、CXCL12对接受一氧化氮吸入治疗PPHN患儿临床转归的预测价值。结果死亡组患儿血清CXCL8、CXCL12水平显著高于存活组(P均<0.05)。多因素Logsitic回归分析结果显示,血清CXCL8水平升高、血清CXCL12水平升高、早产、出生时Apgar评分0~3分、合并并发症是接受一氧化氮吸入治疗的PPHN患儿死亡的危险因素,肺表面活性物质应用、吸入一氧化氮早期反应则是保护因素(P<0.05)。ROC曲线分析结果显示,血清CXCL8、CXCL12联合检测对接受一氧化氮吸入治疗的PPHN患儿死亡预测的曲线下面积(area under the curve,AUC)为0.828,大于血清CXCL8、CXCL12单独检测(AUC分别为0.762、0.714)。结论PPHN患儿血清CXCL8、CXCL12水平升高与接受一氧化氮治疗的不良临床转归有关,且CXCL8、CXCL12水平升高是PPHN患儿死亡的危险因素。CXCL8、CXCL12联合检测对接受一氧化氮治疗PPHN患儿死亡具有较高的预测价值。
文摘目的:分析趋化因子受体8(C⁃C motif chemokine receptor 8,CCR8)在卵巢癌肿瘤浸润性调节性T细胞(regulatory T cell,Treg)中的表达,探讨CCR8对Treg分化的作用。方法:构建C57BL/6小鼠卵巢癌细胞ID8荷瘤模型;流式细胞术检测小鼠肿瘤组织、脾脏和外周血中Treg上CCR8的表达比例,CCR8^(+)Treg上免疫检查点相关蛋白程序性细胞死亡蛋白1(programmed cell death protein 1,PD⁃1)、细胞素性T淋巴细胞抗原4(cytotoxic T⁃lymphocyte antigen 4,CTLA⁃4)、可诱导的T细胞共刺激分子(inducible T cell costimulators,ICOS)、淋巴细胞激活基因3(lymphocyte activation gene 3,LAG⁃3)的表达;流式细胞术检测CCR8变构抑制剂AZ084加入前后对C57BL/6小鼠脾脏中初始CD4^(+)T细胞向Treg分化的影响。结果:卵巢癌荷瘤小鼠肿瘤中Treg上的CCR8表达相比脾脏、外周血的Treg显著增高;相比CCR8^(-)Treg,CCR8^(+)Treg上免疫检查点相关蛋白表达更高;AZ084有效抑制小鼠脾脏中初始CD4^(+)T细胞向Treg的分化。结论:CCR8^(+)Treg在肿瘤浸润性Treg中占主要比例,CCR8作为卵巢癌浸润性Treg的主要标志物,变构CCR8蛋白可以抑制Treg的分化。靶向消除CCR8^(+)Treg可为改善卵巢癌肿瘤微环境的免疫抑制状态提供新思路。
