SIRT2的亚细胞定位及其对293T细胞增殖的影响

目的:构建SIRT2的真核表达载体,并检测其在人胚肾细胞株293T中的亚细胞定位以及对293T细胞增殖的影响。方法:以SIRT2-p IRES质粒为模板PCR扩增获得SIRT2基因目的片段,然后将其克隆到真核表达载体p EGFP-C2中,使目的基因能够和GFP融合表达;采用脂质体法转染293T细胞,在荧光显微镜下观察融合蛋白的亚细胞定位;用核浆分离和Western印迹方法检测融合蛋白的亚细胞定位,加入出核抑制剂细霉素B,进行同样的处理;采用MTT法检测SIRT2对293T细胞增殖的影响。结果:SIRT2主要分布在细胞质中,加入细霉素B后,在细胞质和细胞核中都有明显分布;SIRT2在细胞质中时抑制293T细胞的增殖,当其入核时抑制作用减弱。结论:SIRT2具有核质穿梭功能,它的亚细胞定位是个动态变化过程;SIRT2抑制细胞的增殖,抑制作用的强弱与其亚细胞定位有关。     

AUF1在胞质DNA抑制细胞葡萄糖代谢中的作用研究

目的 探讨AUF1在胞质DNA引起的细胞葡萄糖代谢应答中的作用及其机制。方法 (1)用核质分离技术分离细胞核与细胞质,并通过生物素-亲和素亲和层析技术分离细胞质中与胞质DNA(ISD)结合的蛋白质,然后通过“银染-质谱”和“复合物-质谱”技术鉴定出差异蛋白——AUF1。再利用体外结合实验验证AUF1与胞质DNA的相互作用。(2)在胞质DNA刺激后,通过ATP检测试剂盒和CCK8细胞氧还活力检测试剂,比较野生型细胞和基于CRISPR/Cas9技术的AUF1基因敲除细胞中葡萄糖代谢应答情况。(3)通过半定量PCR技术,在野生型、基因敲除AUF1、基因敲除后回补AUF1或空载体的四类细胞中检测葡萄糖转运蛋白GLUTs以及葡萄糖代谢相关酶的mRNA表达情况,筛选出与细胞糖代谢相关的AUF1下游效应分子——GLUT3。进而用实时荧光定量PCR进行验证。(4)通过半定量和荧光定量PCR分析胞质DNA刺激下GLUT3的mRNA变化情况,分析胞质DNA的刺激是否影响GLUT3的mRNA表达。结果 (1)两次质谱分析均发现AUF1能与ISD结合。体外结合实验也证实,不论是原核表达的GST-AUF1还是真核细胞表达的GFP-AUF1均能与单链和双链的ISD相结合。(2)基因敲除AUF1后的HEK293细胞在用胞质DNA刺激后,胞内的ATP水平和对CCK8的还原能力都明显高于野生型细胞。提示AUF1基因敲除细胞内的葡萄糖代谢不受胞质DNA刺激所抑制,说明AUF1很可能参与了胞质DNA对细胞糖代谢的调节。(3)半定量PCR技术检测发现在AUF1敲除的细胞中GLUT3的mRNA明显减少,而其他的GLUT家族成员和代谢酶则没有显著差异。实时荧光定量PCR证实上述现象,提示AUF1很可能通过稳定GLUT3的mRNA参与葡萄糖代谢的调节。(4)无论是单链还是双链ISD刺激后的细胞中,GLUT3的mRNA均减少,说明GLUT3可能是胞质DNA对糖代谢的调节过程中的一个下游效应分子。结论 AUF1能与胞质DNA结合,很可能通过调节下游GLUT3的mRNA稳定性参与胞质DNA引起的糖代谢应答反应。     

MicroRNA-29b过表达慢病毒载体的构建及其生物学特性的研究

目的:构建针对小鼠microRNA-29b过表达的慢病毒载体,研究其在小鼠神经元GT1-7细胞系中的生物学特性。方法:化学合成两条寡聚核苷酸单链,通过搭桥互补延伸成DNA双链,形成miR-29b的前体结构,将酶切后的慢病毒载体FUGW通过同源重组的方法与miR-29b的前体结构进行连接,构建相应microRNA-29b过表达慢病毒载体,并包装成病毒颗粒后转染小鼠神经元细胞系GT1-7,通过博来霉素药物筛选获得稳转株,RT-PCR检测相关基因在mRNA转录水平上表达量情况。结果:测序图谱证实重组慢病毒表达质粒f-F-miR-29b构建成功,GT1-7细胞稳转株中,miR-29b的表达量与对照组相比提高了约28倍,其靶基因DCX,Vdac1,Pten的表达量有所抑制,性发育相关基因LH-β,kiss-1,Inshulin,IGF-I,GPR54,GnRH,leptin-R没有明显变化。结论:利用慢病毒筛选的方法,成功在小鼠神经元GT1-7细胞中获得microRNA-29b过表达稳转株,为以后microRNA-29b的生物学特性的研究奠定了基础。     

奶牛SCAP基因的克隆,表达定位及对FASN基因启动子的转录调控

固醇调节元件结合蛋白裂解激活蛋白(srebpcieavage activating protein,SCAP)是细胞脂肪合成酶的表达调控因子。本研究旨在肝脏L02细胞中研究SCAP对FASN基因启动子的转录调控作用,有助于进一步明确SCAP/SREBP1对于靶基因的转录调控机制。本研究以荷斯坦奶牛组织c DNA为模板,克隆SCAP基因的编码序列,构建pc DNA3.1-SCAP表达载体,将SCAP表达载体转染L02肝脏细胞,采用荧光定量PCR检测SCAP基因m RNA在24 h、48 h、72 h的表达情况;采用免疫荧光的方法对SCAP标记,激光共聚焦观察SCAP蛋白的亚细胞定位;构建FASN基因启动子载体,转染至肝脏细胞,并分别转染pc DNA3.1-SREBP1和pc DNA3.1-SCAP作为处理,荧光素酶报告基因系统分析对FASN启动子活性的影响。结果发现克隆得到SCAP的PCR产物为3 836 bp的片段,通过与pc DNA3.1载体重组后获得pc DNA3.1-SCAP表达载体,经酶切和测序鉴定正确;将pc DNA3.1-SCAP载体转染肝脏细胞培养24 h、48 h、72 h后,Real-time PCR检测发现与对照组相比,48 h时细胞中SCAP基因m RNA的表达倍数增加了21倍(p0.001);激光共聚焦观察发现,DAPI染色的细胞核呈蓝色,免疫荧光标记的SCAP呈红色,二者融合后发现SCAP定位于细胞质;启动子活性检测发现,与对照组相比,SREBP1质粒处理的细胞FASN启动子活性增加了4.1倍(p0.001),而SCAP和SREBP1共同处理细胞FASN启动子活性极显著增加了7.4倍(p0.000 1)。试验克隆构建奶牛SCAP基因表达载体,亚细胞定位SCAP蛋白主要位于肝脏细胞质中,表明SCAP可以通过结合SREBP1促进FASN基因启动子的转录。     

肝脏特异性CD36基因敲除小鼠的制备及鉴定

目的:运用Cre/Loxp重组酶系统构建肝脏特异性CD36基因敲除小鼠并进行鉴定和验证,为研究CD36的生物学功能奠定基础。方法:构建CD36打靶载体,电转转染胚胎干细胞,通过长链PCR筛选出正确同源重组的阳性克隆,阳性胚胎干细胞克隆经扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合小鼠,再与Flp小鼠交配筛选获得Flox杂合子小鼠,该小鼠与引进的Alb-Cre小鼠交配,在F3代获得CD36fl/fl:Alb-Cre+基因型小鼠,即为肝脏特异性CD36敲除小鼠。采用PCR鉴定小鼠基因型,PCR、实时荧光定量PCR和Western blot验证小鼠肝脏CD36敲除效果,Western blot检测小鼠肾脏、脂肪和心肌组织CD36表达情况,HE染色观察小鼠肝脏形态学改变。结果:建立了CD36基因的Flox杂合子小鼠,与Alb-Cre小鼠交配后,在F3代筛选出CD36fl/fl:AlbCre-和CD36fl/fl:Alb-Cre+基因型小鼠,DNA水平证实CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36基因通过Cre/Loxp重组酶系统被敲除。与CD36fl/fl:Alb-Cre-基因型小鼠相比,CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36mRNA和蛋白表达水平显著降低,肾脏、脂肪和心肌组织CD36蛋白表达无差别,肝脏形态学特征无明显差异。结论:通过Cre/Loxp重组酶系统成功构建了肝脏特异性CD36基因敲除小鼠,为研究CD36在肝脏代谢和肝脏疾病中的功能提供了动物模型。     

小鼠2-细胞胚胎ATP合成酶6基因特异表达分析及鉴定

合子基因组活化是小鼠胚胎早期发育由细胞质调控向核调控转变的关键 .小鼠合子基因组活化发生在 2 细胞胚胎阶段 ,通过对 2 细胞胚胎阶段特异性表达基因的分析 ,可以从分子水平上揭示早期小鼠胚胎的发育机理 .用DD RTPCR技术 ,从单个小鼠 2 细胞胚胎与成熟卵母细胞 (MII细胞 )中分离了 2个差异片段 ,片段 2同小鼠睾丸中表达的一个未知片段具有高度同源性 .经过cDNA文库构建、筛选 ,分离到其全长cDNA .序列分析结果表明 ,该基因为小鼠ATP合成酶亚单位 6基因 .ATP合成酶亚单位 6基因由线粒体DNA编码 ,与细胞内ATP的合成相关 .小鼠 2 细胞胚胎特异表达的ATP合成酶亚单位 6基因可能与胚胎正常发育相关     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。     

EphA3基因启动子区域定位表达及活性分析

目的构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法以Balb／C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL—CMV内对照质粒共转染293T和MEF细胞,分析不同长度的OhA3基因启动子片段的转录活性。结果酶切和测序鉴定表明表达载体构建成功,EphA3基因的核心启动子区域位于-279bp～＋110bp之间,在293T细胞和MEF细胞中其转录活性相似。结论成功构建了荧光素报告基因重组质粒,并确定了BphA3基因的核心启动子区域。     

与衰老相关的线粒体功能严重缺陷的胞质杂合细胞的分子特征

该研究组先前建立了含有不同年龄组小鼠神经细胞线粒体的胞质杂合细胞株(其细胞核基因组背景相同),发现老年组较年轻组的胞质杂合细胞线粒体总体功能显著降低。为了研究与衰老相关的线粒体功能严重缺陷的胞质杂合细胞的分子特征,该研究采用3个线粒体总体功能极其低下的老年组胞质杂合细胞株(1个正常功能年轻组胞质杂合细胞株作为对照)作为研究对象。首先,证实氧耗水平和ATP合成显著降低(P0.05或P0.01)。然后,对线粒体DNA(mitochondrial DNA,mt DNA)进行了高通量测序,特别是检出突变型比例低的mt DNA异质性突变,并进一步对呼吸链复合体依赖性氧耗进行了检测。测序结果显示,在老年组胞质杂合细胞中mt DNA点突变明显积累。这些突变包括非编码区的3个变异,据DNA保守性分析结果,其中2个(异质性m.15849GT、m.16289AT)可能为有害的;编码区的4个变异,据DNA和氨基酸保守性分析及蛋白质功能预测结果,其中2个(ND5基因的同质性m.12496TC、Cyt b基因的异质性m.15199AT)可能为有害的。进一步研究结果显示,同时具有复合体I亚基ND5(或复合体III亚基Cyt b)突变和2个调控区突变的胞质杂合细胞,其复合体I(或复合体III)依赖性呼吸显著降低(P0.05或P0.01)。综上,发生于老年组胞质杂合细胞的线粒体总体功能异常,其原因可能为,mt DNA调控区和编码区的异质性和同质性突变,以及多重mt DNA突变的累加引起线粒体呼吸链复合体功能的缺陷,进而导致线粒体总体功能异常,从而促进衰老。     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

Nucleocytoplasmic transport under stress conditions and its role in HSP70 chaperone systems

BackgroundIn eukaryotic cells, molecular trafficking between the nucleus and cytoplasm is a highly regulated process related to cellular homeostasis and cellular signaling. However, various cellular stresses induce the perturbation of conventional nucleocytoplasmic transport pathways, resulting in the nucleocytoplasmic redistribution of many functional proteins.Scope of reviewWe describe the recent insights into the mechanism and functions of nuclear import of cytosolic chaperone HSP70 under stress conditions and the cellular distribution and functions of its co-chaperones.Major conclusionsHikeshi mediates the nuclear import of the molecular chaperone HSP70. A few of the regulators of the HSP70 chaperone system also accumulate in the nucleus under heat stress conditions. These proteins function collaboratively to protect cells from stress-induced damage and aid in the recovery of cells from stress.General significanceStudies on the regulation of nucleocytoplasmic transport under several cellular stresses should provide new insights into the fundamental principles of protein homeostasis (proteostasis) in both compartments, the nucleus and cytoplasm.     

GTP酶Ran在细胞核运输、核分裂与重建中的作用

小分子的单体G蛋白Ran具有鸟苷三磷酸酶活性,其结合形式Ran-GTP作为区分间期细胞的核质和胞质的一个分子标记,并参与调控核质运输、指导纺锤体形成以及引导核膜解体与装配。现就Ran在真核细胞核质运输、有丝分裂纺锤体组装与核膜动力学中的功能作一综述。     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p,a nucleocytoplasmic protein whose stability depends on its subcellular localization

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.     

异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

目的 研究异源(猪)基因α1,3半乳糖转移酶(3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响.方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体p...     

Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading

The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.