Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells

BACKGROUND:Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells.Previously,we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase(hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I(hArgI) and human ornithine transcarbamylase(hOTC) in pathway 2.The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells.METHODS:We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line.The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods.Cell cycle PCR chip was used to assess the changes of gene expression.RESULTS:Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression,but inhibited hArgI expression.The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate(Glu) and with 60 and 180 mmol/L NH 4 Cl,respectively.In addition,the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day,indicating that introduction of hGS impedes HepG2 cell proliferation.Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role.CONCLUSIONS:This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism.The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

瞄准：用侵入人体气管粘膜的病菌向量为可溶的 SARS S 蛋白质的准备开发一个高度有效的方法为 S 蛋白质调查满足要求。方法：人的侵入人体气管粘膜的病菌向量被用来表示可溶的 S 蛋白质(相应于 1 约 1190 氨基酸) 在 HEK239 房间用优化 codon 的基因构造与 Myc/His 熔化了标签。忍受 S 蛋白质基因的 recombinant 侵入人体气管粘膜的病菌被结扎方法产生。有 Myc/His 标签的表示 S 蛋白质与统帅玫瑰祷告由分离跟随了的 Ni-NTA 从培养基被净化。S 蛋白质被西方的污点检测，它的生物学的活动被绑定分析到 Vero 房间。结果：在感染剂量(50 的 MOI ) 和表示时间(48 h ) 的条件下面， S 蛋白质的高级表示被获得。表示水平决心是在纯化以后的约 75 个 microg/10 (6 ) 房间。净化的可溶的 S 蛋白质被西方的污点乐意地与 anti-Myc 抗体检测并且显示了能力绑 Vero 房间出现，证明可溶的 S 蛋白质能仍然是在本国的分子的生物学的活动。结论：在 HEK293 房间的 S 蛋白质的高级表示由侵入人体气管粘膜的病菌调停了能在优化表示条件下面被完成。蛋白质拥有生物学的活动，它为 S 蛋白质生物功能的进一步的调查打一个基础。