Cell surface activation of progelatinase A （proMMP—2） and cell migration

Gelatinase A (MMP-2) is considered to play a critical role in cell migration and invasion.The proteinase is cerceted from the cell as an inactive zymogen.In vivo it is postulated that activation of progelationase A (proMMP-2) takes place on the cell surface mediated by membrane-type matrix metalloproteinases (MT-MMPs).Recent studies have demonstrated that proMMP-2 is recruited to the cell surface by interacting with tissue inhibitor of metalloproteinases-2 (TIMP-2) bound to MT1-MMP by forming a ternary complex.Free MT1-MMP closely located to the ternary complex then activates proMMP-2 on the cell surface.MT1-MMP is found in cultured invasive cancer cells at the invadopodia.The MT-MMP/TIMP-2/MMP-2 system thus provides localized expression of proteolysis of the extracellular matrix required for cell migration.     

cAPK催化亚基的C端融合表达,纯化及其NMT底物活性作用

将小鼠ｃＡＭＰ依赖蛋白激酶催化亚基α（ｍＣα）基因从３＇端截去９６ｂｐ后与Ｈｉｓ－ｔａｇ融合,构建Ｃ端融合Ｈｉｓ６的表达质粒ｐＺＰｍＣα４Ｈ。该质粒在大肠杆菌ＢＬ２１（ＤＥ３）中得到了高效表达,目的蛋白ｍＣα４Ｈ的表达量与ｍＣα相近。该蛋白在３７℃表达时主要以包含体形式存在,而２２℃表达时则大部分可溶。利用固定化金属离子（Ｎｉ＾２＋）配体亲和层析,分别从破菌上清涂和包含体中纯化ｍＣα４Ｈ,结果表明     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

非受体酪氨酸蛋白激酶在病毒与宿主相互作用过程中的作用

非受体酪氨酸蛋白激酶（ＮＲＰＴＫ）与调节细胞生长,分化与成熟过程的信号传导途径密切相关,近年来发现疱疹病毒科,逆转录病毒科和乳多空病毒科病毒的蛋白质能与ＮＲＰＴＫ发生作用,并证实这类无关的病原体却具有相似的干扰细胞蛋白质的机制。     

Immunoelectron Microscopy for Locating Calvin Cycle Enzymes in the Thylakoids of Synechocystis 6803

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.     

The Chloroplast Kinase Network： New Insights from Large-Scale Phosphoproteome Profiling

Protein phosphorylation is one of the most important posttranslational modifications in eukaryotic cells and affects almost all basic cellular processes. The chloroplast as plant-specific cell organelle with important metabolic functions is integrated into the cellular signaling and phosphorylation network. Recent large-scale chloroplast phosphoproteome analyses in Arabidopsis have provided new information about phosphorylation targets and expanded the list of chloroplast metabolic and regulatory functions that are potentially controlled by protein phosphorylation. Phosphorylated peptides identified from chloroplast proteins provide new insights into phosphorylation motifs, protein kinase activities, and substrate utilization. Phosphorylation sites in protein kinases can reveal chloroplast phosphorylation cascades that may network different functions by integrating signaling chains. Our review provides a meta-analysis of currently available chloroplast phosphoproteome information and discusses biological insights from large-scale chloroplast phosphoprotein profiling as well as technological constraints of kinase network analysis.     

PEI-CS/siRNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响

目的:探讨聚乙烯亚胺-壳聚糖(PEI-CS)/si RNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响。方法:采用复凝聚法将PEI-CS(100μg/m L)与不同浓度的MRE11 si RNA-FAM形成PEI-CS/si RNA复合颗粒,并转染BEL7402/5-FU细胞,用荧光显微镜和Real-time PCR检测转染效率和沉默效率。结果:荧光显微镜观察结果显示:转染细胞48 h后,3.125、6.25、12.5、25、50μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为62.31%、76.09%、79.99%、86.49%、96.59%。转染细胞48、72、96 h后,12.5μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为78.22%、55.76%、42.85%,25μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为83.67%、74.23%、67.45%。Real-time PCR检测结果显示:25μg/m L的si RNA与PEI-CS形成的复合颗粒转染48小时后,对BEL7402/5-FU细胞中MRE11基因的沉默效率为35.4%。结论:聚乙烯亚胺-壳聚糖/si RAN复合颗粒能有效转染肝癌耐药细胞Bel7402/5-FU,并对BEL7402/5-FU细胞中MRE11基因表达有一定抑制作用。     

细胞对机械刺激感知的生物力学机制

细胞作为机体的基本单位,始终处于一个受力环境中。微环境中的细胞不仅受化学信号的影响,还受基质刚度、外界力载荷及胞外基质(ECM)结构的调控,这些都能影响细胞分化、迁移、形态发生、增殖等方面的生物学反应。目前,部分学者致力于细胞生物力学的应用和机制方面的探索,但对于细胞力学感知机理的认知仍无法形成一个完整的定论。本文将就整合素、G蛋白耦联受体、张力活化通道(SAC)、细胞核等介导的细胞生物力学感知通路作一综述。     

ACE、CYP11B2基因多态性与慢性心力衰竭患者醛固酮脱逸的关系

目的:研究CYP11B2-344C/T(醛固酮合成酶)及ACEI/D(血管紧张素转化酶)基因多态性与慢性心力衰竭(CHF)患者实施ACEI治疗后出现醛固酮脱逸表现的关系。方法:回顾分析2008年10月至2012年10月我科收治的252例CHF患者,全部患者应用ACEI治疗3月,醛固酮在基线以上为醛固酮脱逸,依据此标准将患者分为研究组(脱逸组,n=86)与对照组(非脱逸组,n=166),依据PCR(聚合酶链反应)及RFLP(片段长度限制多态性)等方法分别检测两组CYP11B2及ACE基因型,比较两组基因型频率的分布。结果:252例患者中,共86例出现醛固酮脱逸,发生率为34.1%。全部受试患者CYP11B2基因型及ACE基因型频率与Weinberg-Hardy平衡均相符(P均0.05)。研究组ACE I/D三种基因型的组间分布与对照组相较,无统计学差异(P0.05);CYP11B2基因TT型的频率与对照组相较,呈明显统计学差异(P0.05),等位基因C/T频率的组间分布同对照组相较,亦呈明显差异(P0.05)。研究组ACEI/D的基因多态性及CYP11B2-344C/T的多态性中,基因型联合组间分布与对照组相较,无统计学差异(P0.05)。结论:ACE基因多态性与CHF患者ACEI治疗后出现醛固酮脱逸无关,CYP11B2基因T等位基因及TT基因型多态性可能是CHF患者ACEI治疗后发生醛固酮脱逸的高危因素。醛固酮脱逸时,ACE、CYP11B2基因不具有协同效果。     

革兰氏阴性菌的Cpx双组分调控系统

细菌主要通过单组分、双组分、三组分调控系统来适应外界环境变化以保证自身的正常生长和繁殖。Cpx系统是革兰氏阴性菌中普遍存在的双组分调控系统之一,作为双组分调控系统的重要一元,它由细胞膜组氨酸蛋白激酶CpxA、细胞质响应调节蛋白CpxR以及细胞周质空间辅助调节蛋白CpxP构成。本文着重介绍了Cpx系统各组分的结构特征,并结合前人的文献和我们近期的研究成果,综述了Cpx系统信号整合的最新研究进展,提出了尚待解决的问题及进一步的研究方向,为科研人员对Cpx双组分调控系统的研究提供一定的帮助。