严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

Extracellular ATP and adenosine modulate tumor necrosis factor-induced lysis of L929 cells in the presence of actinomycin D

Extracellular ATP in concentrations of 0.5 to 2.5 mM modulates TNF-induced cytolysis of L929 cells in the presence of actinomycin D. When present throughout the entire assay period, it inhibits the TNF-induced cytolysis. ADP was less active whereas AMP and GTP were nonreactive. However, inhibition was also achieved by adenosine that was nearly as active as ATP. Yet, the inhibitory effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine. Thus, the nonhydrolyzable ATP analogue adenyl(beta-gamma-methylendiphosphate) was equally effective in inhibiting TNF-induced cytolysis. Moreover, no conversion of ATP into adenosine was observed during the entire assay period. However, inhibition no longer occurred when the TNF and ATP containing medium was removed after 5 h and replaced by a fresh medium containing TNF and no ATP. We now observed substantial enhancement of the TNF-induced cytolysis by ATP. Finally, treatment with N6-(R-phenylisopropyl)adenosine or with aminophylline, which are thought to downregulate adenosine receptors and to prevent binding of ligands to adenosine receptors, respectively, abolishes adenosine and ATP-mediated inhibition. Again, substantial enhancement of the TNF-induced cytolysis was observed by ATP and only a minor effect by adenosine. The results together suggest that ATP interacts with purinoceptors on the plasma membrane and is capable to enhance and inhibit TNF-induced cytolysis under appropriate conditions. The outcome of the ATP-induced modulation may be influenced by adenosine receptors.     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.