细胞表面半乳糖基转移酶及其生物学功能

根据mRNA转录子的大小,β-1,4-半乳糖基转移酶分为短型和长型两类半乳糖基转移酶.短型的位于高尔基体的成熟面.长型的主要表达在细胞表面,通过与相邻细胞表面或细胞外基质上的适当的糖苷底物的结合介导细胞-细胞和细胞-基质间的相互作用,如精子发生、精卵结合、早期胚胎细胞间粘附、次生滋养层巨细胞迁移和神经轴突向外生长等,或作为胞外寡糖链配基的信号传递受体影响G蛋白信号途径.另外,表面半乳糖基转移酶通过调节表皮生长因子受体信号传导能力向胞内传递生长抑制信号,在细胞增殖控制中起重要作用.     

反义RNA对猪α-1,3-半乳糖苷转移酶活性的影响

 α 1,3 半乳糖表位是猪 人异种移植超急性排斥反应的主要抗原 ,由α 1,3 半乳糖苷转移酶催化合成 .用RT PCR方法扩增中国实验用小型猪α 1,3 半乳糖苷转移酶cDNA的前 582bp ,测定碱基序列并构建其反义表达载体pLXRN ,将其转染入猪主动脉内皮细胞 .NorthernBlotting表明α 1,3 半乳糖苷转移酶mRNA减少 .检测α 1,3 半乳糖苷转移酶活性表明 ,反义RNA可使其活性下降32 2 % .研究结果表明可能通过反义RNA来抑制猪 人异种移植超急性排斥反应     

蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析

【目的】蔗糖:蔗糖-1-果糖基转移酶催化1分子蔗糖上的果糖基转移到另一个蔗糖分子上,形成1-蔗果三糖和葡萄糖。在低聚果糖中,1-蔗果三糖益生素活性最高。本研究将该酶展示在酵母菌细胞表面上,并用于1-蔗果三糖的制备。【方法】将来自莴苣的蔗糖:蔗糖-1-果糖基转移酶基因克隆到用于酵母细胞表面展示的表达载体上,并在解脂亚罗酵母菌中进行异源表达,表达的酶展示在该细胞表面上,然后以蔗糖为底物,研究表面展示的蔗糖:蔗糖-1-果糖基转移酶的性质。【结果】免疫荧光实验结果表明蔗糖:蔗糖-1-果糖基转移酶已展示在酵母菌的细胞表面上,高效液相色谱结果表明酵母表面展示的该酶具有转移酶的催化活性。该酶的最适作用温度、最适作用p H分别为45°C和7.5;该酶的催化活性受Zn2+和Cu2+的抑制,受Ca2+激活;该酶重复使用7次后,酶活下降50%。表面展示的蔗糖:蔗糖-1-果糖基转移酶和3%蔗糖混合后在40°C条件下孵育30 min后,所产1-蔗果三糖含量最高为20.8 mmol/L。【结论】蔗糖:蔗糖-1-果糖基转移酶在解脂亚罗酵母菌中得到成功表达,并展示在其细胞表面上,生化研究表明该重组蛋白具有果糖基转移酶活性,且催化蔗果三糖的生成。表面展示的蔗糖:蔗糖-1-果糖基转移酶作为一种全细胞催化剂能够用于1-蔗果三糖的制备。     

细胞诱导分化剂对HL60细胞β-1,4-半乳糖基转移酶活性的影响

用荧光标记的受体底物（Ｇｎβ１－２Ｍα１－６（Ｇｎβ１－２Ｍα１－３）Ｍβ１－４Ｇｎβ１－４（Ｆｕｃα１－６）Ｇｎ－ＰＡ）,结合高效液相层析（ＨＰＬＣ）建立了细胞β－１,４－半乳糖基转移酶的活性检测方法．研究了ＨＬ６０细胞在体外低血清培养后不同的时间其酶活性的变化,发现１２至２４ｈ酶活性达到一个高峰,为５０．１４ｐｍｏｌ／ｍｉｎ（１０６ｃｅｌ）,此时细胞处在分裂间期,其它各测定时间变化不大．用ＰＭＡ,ＲＡ等细胞诱导分化剂处理ＨＬ６０细胞株时,发现其活性发生了较明显的变化,ＰＭＡ诱导的细胞其酶活性在２４ｈ变化最大,升高到对照的１．３２倍;而ＲＡ处理的细胞其酶活性在７２ｈ变化最大,升高到对照的２．１５倍．     

β-半乳糖苷酶基因在猕猴桃果实成熟过程的表达

从成熟中华猕猴桃果实中克隆到了一个 β 半乳糖苷酶基因cDNA片段 ,其长度为 747bp ,有一由 2 49个氨基酸组成的开放阅读框架 ,它与苹果、芦笋、绿花椰菜、番茄中 β 半乳糖苷酶基因cDNA相应区段的核苷酸同源性为 6 7.3 %～ 70 .3 %,氨基酸同源性为6 9.1%～ 72 .7%。用该片段为探针进行Northern分析表明 ,果实采收时 ,β 半乳糖苷酶mRNA水平最高 ,随后呈下降变化 ,同时 β 半乳糖苷酶基因的表达可为外源乙烯所诱导 ,但在果实乙烯跃变期间 β 半乳糖苷酶基因的表达信号无显著变化。文中对 β 半乳糖苷酶在猕猴桃果实成熟衰老过程的作用进行了讨论     

eIF4E作用及调节机制

eIF4E作为翻译启动因子复合体eIF4-F的关键部分,调节细胞的蛋白质合成的限速步骤,ras基因加强eIF4E的磷酸化,c-myc基因增加eIF4E mRNA水平,eIF4E活性随其本身磷酸化的加强而加强,随其结合蛋白的磷酸化加强而减弱。eIF4E是新近发现的原癌,这量的eIF4E选择性地加强原癌基因等“弱mRNA”的翻译：可以通过加强蛋白质合成的速度,也可以加速mRNA由细胞核由胞浆转移。     

硒性白内障大鼠模型晶状体中GR和GSH-Px的表达

 为探讨硒性白内障大鼠晶状体中谷胱甘肽过氧化物酶 (GSH Px)和谷胱甘肽还原酶 (GR)的活性调节在硒性白内障形成中的作用及调节方式 ,采用半定量RT PCR方法 ,比较正常晶状体、核中心混浊晶状体 (核白 )和完全混浊晶状体 (全白 )中GSH Px和GR的mRNA水平及酶活性的变化 .研究发现 ,核白晶状体中 2种酶的活性和mRNA水平均升高 ,其中酶活性的升高幅度小于mRNA水平 .随着白内障的发展 ,2种酶的活性和mRNA水平均逐渐下降 .至晶状体全白时 ,2种酶的活性均显著低于正常 ;全白时GR的mRNA水平降至正常 ,GSH Px的mRNA水平则仍高于正常 .结果表明 ,硒性白内障形成与细胞内GSH Px和GR的活性调节密切相关 ,GSH Px和GR的活性调节可能主要发生在转录水平     

慢病毒介导的小鼠PINK1基因RNAi载体的构建及在NIH3T3细胞中的筛选

目的:构建筛选靶向特异PINK1-siRNA慢病毒表达载体,感染小鼠胚胎细胞(NIH3T3)验证该病毒载体的敲减效率,为研究帕金森病的发病机制奠定基础。方法:构建2对靶向小鼠PINK1-siRNA序列(KD1和KD2),将这2对序列连接在GV118上,将重组载体和病毒包装的辅助质粒共转染293 T细胞,获得慢病毒颗粒,再将该病毒颗粒转染入NIH3T3细胞,用qRT-PCR验证细胞内PINK1 mRNA表达水平以验证敲减效果。结果:筛选出可以用于后续实验的高效靶向KD2序列,并成功转染到小鼠NIH3T3细胞中,在感染复数(MOI)为50时,KD2的沉默效果最好,敲减率达到66.4%。结论:成功构建了高效靶向小鼠PINK1-siRNA慢病毒载体,其可稳定转染小鼠NIH3T3细胞,可高效抑制PINK1 mRNA的表达。为下一步利用其感染神经细胞或注入动物脑内,进一步研究PINK1基因在帕金森病发病过程中的作用环节和机制提供了分子生物学的技术基础。     

1,6-二磷酸果糖与细胞保护

1,6-二磷酸果糖是细胞内糖代谢的中间产物,是能在分子水平上调节细胞代谢中若干酶活性,作为恢复和改善细胞代谢的药物,可通过多方面因素减轻细胞损伤,从而对细胞起保护作用。     

α-硫辛酸对缺氧应激肝癌细胞线粒体呼吸率和产能代谢的影响

通过实验阐明抗氧化剂α-硫辛酸（alpha-lipoic acid,α-LA）对肝癌细胞内活性氧具清除作用,并发现其对肝癌细胞和正常肝细胞增殖有不同作用影响。在缺氧条件下,研究使用抗氧化剂干预对肝癌细胞和正常肝细胞缺氧耐受性,线粒体活性和产能代谢的影响及差异。以SMMC-7721人肝癌细胞和L02正常肝细胞作为研究对象,在α-硫辛酸干预条件下检测细胞生长曲线和细胞内ROS;分别在单纯缺氧及加α-硫辛酸缺氧条件下,检测细胞存活率、细胞内ROS、细胞耗氧率、细胞生成ATP和癌基因c-myc mRNA的表达。实验结果说明：缺氧情况下,肝癌细胞通过增加糖酵解途径的产能方式诱导ATP能量代偿能力提高。使用抗氧化剂α-硫辛酸干预清除细胞内过剩ROS,能降低肝癌细胞线粒体呼吸率,并能通过下调c-myc表达抑制肝癌细胞的增殖及降低其缺氧耐受性。     

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.     

Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.     

In Vitro transformation of LW13 Rat liver epithelial Cells

A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.     

Analysis of ras oncogene products by two-dimensional gel electrophoresis: evidence for protein families with distinctive molecular forms

Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.     

维甲酸对鼻咽癌细胞生长、表型和瘤基因表达的作用

研究了维甲酸（ＲＡ）对鼻咽癌细胞生长、表型和癌基因表达的作用．用ＲＡ诱导鼻咽癌细胞,绘制诱导前后的细胞曲线,观察细胞形态,并用Ｎｏｒｔｈｅｒｎ杂交和ＤＮａｓｅⅠ超敏感区分析法检测基因表达和调控．结果表明,ＲＡ能显著抑制鼻咽癌细胞的生长,前５ｄ下降约５０％．ＲＡ处理后的细胞从典型的多边形形态变成扁平、细长,类似纤维细胞状的形态．ＲＡ诱导前ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因ＨＮＥ２细胞中高表达,而诱导后ｃ－ｍｙｃ基因表达水平急剧下降,ｃ－Ｈａ－ｒａｓ基因无明显改变．在实验中还发现ＲＡ诱导前后的ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因中一些重要的超敏感位点和它们的功能．由实验结果可得到如下结论：ＲＡ能促进鼻咽癌细胞分化,通过对染色体上调控位点的作用来抑制ｃ－ｍｙｃ基因的表达,ＤＮａｓｅⅠ超敏感位点与细胞的分化程度、细胞的组织特异性和基因表达状态有关,ｃ－ｍｙｃ基因可通过不同的调控方式而失活．     

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.     

Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase

Gamete recognition in the mouse is mediated, in part, by the binding of sperm surface galactosyltransferase (GalTase) to appropriate substrates in the egg zona pellucida. In this paper, sperm GalTase is shown to be an externally oriented, integral plasma membrane component. GalTase is not peripherally adsorbed to the cell surface, nor is it bound to cell surface glycoside substrates. GalTase can be released from the surface of intact sperm by either mild proteolysis or by detergent under conditions in which the sperm membranes remain intact as judged by double-label indirect immunofluorescence. Detergent-solubilized sperm GalTase has been purified to apparent homogeneity by affinity chromatography and characterized as a beta 1,4-GlcNAc:GalTase by substrate and kinetic analyses. Purified and membrane-bound GalTase both show an unusual thermal inactivation above 39-40 degrees C, whereas other sperm enzyme activities as well as GalTase activity from other cell types are temperature-dependent. Purified sperm GalTase inhibits sperm binding to the egg zona pellucida, consistent with its proposed role during gamete recognition.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Temporally specific involvement of cell surface beta-1,4 galactosyltransferase during mouse embryo morula compaction

Cell surface beta-1,4 galactosyltransferase (GalTase) is shown to mediate intercellular adhesions between embryonal carcinoma (EC) cells and specifically during late morula compaction in the preimplantation mouse embryo. Monospecific anti-GalTase IgG raised against affinity-purified bovine beta-1,4 GalTase recognizes F9 EC cell GalTase as judged by immunoprecipitation and inhibition of GalTase activity, as well as by immunoprecipitation of a single 52 kd metabolically labeled membrane protein. Anti-GalTase IgG inhibits cell adhesions between EC cells, dissociates compacted mouse morulae, and inhibits blastocyst formation. Anti-GalTase IgG specifically inhibits cell adhesions during late morula compaction, coincident with a peak of surface GalTase activity as determined by direct enzyme assay. On EC cells, GalTase activity can be proteolytically released from intact cells, and is localized by indirect immunofluorescence to areas of intercellular contact, consistent with its proposed role in cell adhesion. Beta-1,4 GalTase is the first cell adhesion molecule identified that participates during late morula compaction, subsequent to uvomorulin function.