FITC标记的GSTP1蛋白在鼠巨噬细胞中的定位

GSTP1(GSTπ)是人体内谷胱甘肽-S-转移酶(GST)的一种重要的活性亚型,属于机体内源性蛋白,不会引起免疫原反应,在维持细胞的正常机能中发挥着重要作用,也是一种具有临床应用价值的抗氧化剂.本实验室在研究中首先发现GSTP1具有明显的抗炎作用,由此我们设想可以通过增加外源性的GSTP1蛋白来提高其在机体内的水平,以此增强机体的抗炎能力.本实验将薄层色谱扫描和SDS-PAGE相结合用于FITC标记蛋白质的检测,使用激光共聚焦显微镜和流式分析相结合的方法研究了外源GSTP1蛋白在RAW264.7巨噬样细胞和鼠腹腔巨噬细胞中的跨膜转位,结果显示外源GSTP1蛋白能够进入上述细胞.该研究为GSTP1蛋白作为直接使用的药物在抗肿瘤、抗炎、抗氧化等领域的应用提供了实验基础.     

巨噬细胞毒素用于降低CBA/J×DBA/2小鼠胚胎吸收率

目的 :研究巨噬细胞毒素降低反复自然流产模型CBA/J×DBA/ 2小鼠胚胎吸收率的免疫机制 .方法 :在小鼠交配前腹腔注射变性蛋白质包被的二氧化硅颗粒 ,做为巨噬细胞毒素以去除巨噬细胞 ,观察胚胎吸收率和淋巴细胞亚群变化 ,进而分析淋巴细胞浸润与妊娠结局的关系 .结果 :二氧化硅预处理组平均每窝活胎数显著增多 (6 3± 1 5vs 3 5± 1 0 ,P <0 0 1) ,胚胎吸收率则显著下降 (18 2 %vs .38 2 %,P <0 0 5 ) .与此相应 ,CD3 CD6 9 、CD3 CD71 细胞比率和CD4 5 MHC -II 、CD80 MHC -II 细胞比率均显著降低 .结论 :体内注射巨噬细胞毒素可以显著降低CBA/J×DBA/ 2小鼠母胎交界面活化型T细胞和某些巨噬细胞亚型的比率 ,并且这种变化与胚胎吸收率的下降相联系 .     

试述鲍类前合子生殖隔离

生殖隔离是一个最重要的生物过程。虽然有许多机理还是未知的,但已研究清楚了物种之间的生殖隔离有两种方式。即通过配子不成熟、不受精的前合子生殖隔离和杂交后代的不育、不成活的后合子生殖隔离。在动物受精过程中,两个配子的质膜在接触融合之前,精子必须穿透包绕在卵细胞外的物质,为完成这一过程,大多数种类的动物都发展了精子结构,使其带有顶体泡,它通过泡吐作用释放具有溶解作用的蛋白质,由它担任穿透卵细胞外被的作用。成熟的鲍精子具有发达的顶体泡,里面含有多种蛋白质,但其中的两种蛋白质－细胞溶素（16kDa）和18kDa蛋白质与生殖有关,是生殖隔离的主要分子结构。当精子粘附到卵黄外膜上时就诱发了顶体反应,释放的16kDa蛋白质利用非酶解反应,在卵黄膜上穿一个小孔,精细胞便从此处穿过卵黄膜与卵细胞融合。鲍类的卵细胞由一层又厚又复杂的细胞外基质包被,并由胶状物、卵黄膜和细胞表面壳等几种成分组成。超微结构研究发现：胶状物位于卵细胞的最外层,由疏松排列的纤维组成,它在鲍类受精中的作用还没有研究不禁。鲍类的ESC层与海胆的卵黄膜十分相似,含有精子结合受体,它是不分支的糖蛋白,同同细胞溶素结合的惟一成分,它具有种属特异性。每个受体大约同60个细胞溶素结合。所以种间受精的抑制因素也许并不只有卵黄膜与溶解蛋白的反应,还可能有精子对ESC层的粘接作用。     

有机磷水解酶全细胞生物催化剂的特性

利用有机磷水解酶大肠杆菌细胞表面展示工程菌为全细胞催化剂,研究了其降解对氧磷的反应.确定了最适反应条件为:反应温度37 ℃、pH值8.0、底物浓度2.0 mmol/L、振荡速率120 r/min,在此条件下反应5 h,对氧磷降解率可达80%左右,且全细胞催化剂具有较好的稳定性和可重复使用性,显示了良好的应用前景.     

采用比较蛋白质组学方法研究p38β激酶对293T细胞蛋白质表达的影响

采用比较蛋白质组学方法研究了过表达p38信号通路激酶p38β对人类肿瘤细胞293T细胞蛋白质表达的影响,利用双向电泳和质谱技术分离鉴定了13个差异表达蛋白质.这些差异蛋白的分布从细胞质膜到核,其功能涉及核酸、蛋白质、糖脂类分子的转录、合成和成熟,细胞物质跨膜运输,细胞周期调控以及细胞防御等.文献分析并没有发现上述鉴定的差异表达蛋白是p38β激酶现有直接底物的证据,提示其可能为新的p38β激酶相关蛋白.这些结果表明,比较蛋白质组学方法是寻找激酶新靶标的一种有效工具.     

污泥混合液对膜生物反应器膜污染影响的研究

考察膜生物反应器处理污水过程中,污泥混合液中以及膜上胞外聚合物(EPS),溶解性微生物产物(SMP)以及其中蛋白质,多糖,蛋白质/多糖(p/c),黏度等对膜污染的影响,进而为膜污染的研究和工程实践提供理论指导.结果表明:膜上EPS以及其中的蛋白质,多糖是考察的多个因素中对膜污染的影响最大的因素.污泥混合液中多糖,p/c,膜上p/c,黏度与TMP的相关性都较大,是对膜污染影响的较大因素.污泥混合液中EPS以及其中的蛋白质,SMP以及其中的蛋白质,多糖和p/c对膜污染的影响较小.与多糖相比蛋白质对膜污染贡献较大.     

线粒体膜间隙蛋白质释放与细胞凋亡研究进展

近年来,国内外有关线粒体和肿瘤的关系研究的越来越多,开发与线粒体相关的新型药物并诱导肿瘤细胞凋亡逐渐成为热点.该文主要综述了近年来关于几种线粒体膜间隙蛋白质在细胞凋亡中的作用和由线粒体中释放的可能机制及其在肿瘤药物治疗中的最新进展,指出线粒体膜间隙蛋白质在诱导肿瘤细胞凋亡中具有重要作用.     

葡萄糖转运体4与Ⅱ型糖尿病相互关系的研究进展

葡萄糖转运体4(GLUT4)是转运葡萄糖的重要蛋白质,与Ⅱ型糖尿病(T2DM)密切相关.从GLUT4对骨骼肌、心肌和脂肪组织,以及肾组织的肾小球系膜细胞的影响等方面进行了综述,研究葡萄糖转运体4与Ⅱ型糖尿病相互关系.     

纳米银生物学效应研究进展

综述了纳米银毒理学和生物效应方面的研究进展.通过分析纳米银的理化特性、进入人体途径以及在呼吸道、皮肤和胃肠道暴露途径下纳米银与组织的相互作用,认为在开发应用纳米银产品的同时更应关注可能产生的负面生物效应,并提供完整且符合实际的毒理学评价资料.国内外学者对纳米银细胞毒性的研究结果显示,纳米银可与蛋白质和酶的巯基发生反应,引起细胞线粒体功能损害、膜渗透及细胞形态的凋亡样变化,其毒性机制尚未阐明,可能是由于氧化应激及脂质过氧化介导所致.因此,在加强纳米银毒理学和生物效应研究的同时,应建立评价纳米产品生物安全性的标准方法及评价体系,关注可能导致的环境问题,深入研究纳米银引起的特殊生物环境效应.     

超声波辐照对大肠杆菌细胞膜的影响

为了解大肠杆菌经超声波作用后其细胞膜的变化状况,采用荧光染料二乙酸荧光素、罗丹明123、二氯荧光黄双乙酸盐对大肠杆菌样品染色,并通过荧光分光光度计、荧光显微镜和透射电镜检测、观察.结果显示:荧光探针测定法可用于测定大肠杆菌细胞膜的变化.在超声波频率为25 kHz、电功率为300W的条件下处理90 s后,细胞结构完整,膜双分子层变模糊,表面有破损成小孔的地方,内含物外渗.膜通透性增加12.7%,膜电位下降26.7%,胞内活性氧水平上升.     

Requirement for c-ras proteins during viral oncogene transformation

Many retroviral oncogenes have been classified into one of several categories based on structure, enzymology and cellular localization. These genes originated from host cells and are probably derived from genes normally involved in the control of cell proliferation. The cellular counterparts of three oncogenes have been identified as a growth factor or growth factor receptor; related oncogenes include receptor-like membrane proteins which often express tyrosine kinase activity. These growth factor-related oncogenes are structurally and biochemically distinct from the membrane-associated ras gene family, which bind and hydrolyse GTP. Oncogenes localized primarily in the cytoplasm which probably have serine kinase activity, have also been identified. Although the structure and biochemistry of many oncogenes have been extensively studied, relatively little is known about the functional relationships of oncogene proteins within the cell. An opportunity to study such interaction is provided by the identification of a monoclonal antibody that neutralizes cellular ras proteins when microinjected into cells. It has been shown previously that the injected antibody inhibits the initiation of S-phase in NIH 3T3 cells. In the present study we injected this monoclonal antibody into NIH 3T3 cells transformed by a variety of oncogenes. The results show that transformation by three growth factor receptor-like oncogenes depends on c-ras proteins, while transformation by two cytoplasmic oncogenes appears to be independent of c-ras protein.     

Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.     

Lectin-like polypeptides of P. falciparum bind to red cell sialoglycoproteins

Attempts to control human malaria by immunological means could be compromised by antigenic variability within and between different strains of malarial parasites1. A useful alternative approach might be to block parasite antigens which are important in the mechanisms of invasion of red cells. As the major human parasite Plasmodium falciparum is highly specific for human red cells, isolation of the proteins involved in the recognition of red cells by this parasite might be of particular value. Recent studies suggest that the major red cell sialoglycoproteins (SGPs), glycophorins A, B and possibly C, may carry the sites recognized by the parasite2-4. Furthermore, because certain carbohydrates present on SGPs such as N-acetylglucosamine are able to block invasion by the parasite5, they may be involved in the initial interaction between parasite and red cell. We have now identified parasite proteins which bind to SGP or N-acetylglucosamine on Sepharose 4B columns. Three proteins, of molecular weights (MWs) 140,000 (140K), 70K and 35K, seem to be specifically bound by N-acetylglucosamine.     

Eph receptors and ephrins restrict cell intermingling and communication.

Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells and neuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. We therefore analysed whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signalling. Here we report that bidirectional but not unidirectional signalling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behaviour that can stabilize a distinct identity of adjacent cell populations.     

A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

To investigate proteins expressed in the renal tissue of the passive Heymann nephritis (pHN) rat model,we prepared pHN rat models with anti-FxA1 serum and analyzed the proteins differentially expressed in the kidney tissue with label-free liquid chromatography-tandem mass spectrometry.We then analyzed in depth the endoplasmic reticulum stress (ERS)-related protein using an online bioinformatics platform.Forty-one differential proteins and their annotations were obtained.Gene Ontology (GO) function analysis showed that 16 proteins were involved in cellular metabolism and 22 were proteins related to catalytic activity,including protein folding or ATPase.Protein-GO networks indicated that VCP could interact with the ERS marker HSPa5,with both involved in a single pathway.On inhibition of podocyte VCP by RNAi under normal conditions,the HSPa5 expression level did not change,but when the cell was subjected to ERS by tunicamycin,HSPa5 expression significantly increased with RNAi of VCP when compared with the tunicamycin-treated group.Our results showed that ERS plays an important role in podocyte injury of membranous nephropathy and is mediated by an HSPa5-VCP signaling pathway,in which the most predominant proteins are those related to cellular metabolism and catalytic activity.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.

14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died ('mitotic catastrophe') as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.     

Rapid neutrophil adhesion to activated endothelium mediated by GMP-140.

Granule membrane protein-140 (GMP-140), a membrane glycoprotein of platelet and endothelial cell secretory granules, is rapidly redistributed to the plasma membrane during cellular activation and degranulation. Also known as PADGEM protein, GMP-140 is structurally related to two molecules involved in leukocyte adhesion to vascular endothelium: ELAM-1, a cytokine-inducible endothelial cell receptor for neutrophils, and the MEL-14 lymphocyte homing receptor. These three proteins define a new gene family, termed selectins, each of which contains an N-terminal lectin domain, followed by an epidermal growth factor-like module, a variable number of repeating units related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Here we demonstrate that GMP-140 can mediate leukocyte adhesion, thus establishing a functional similarity with the other selectins. Human neutrophils and promyelocytic HL-60 cells bind specifically to COS cells transfected with GMP-140 complementary DNA and to microtitre wells coated with purified GMP-140. Cell binding does not require active neutrophil metabolism but is dependent on extracellular Ca2+. Within minutes after stimulation with phorbol esters or histamine, human endothelial cells become adhesive for neutrophils; this interaction is inhibited by antibodies to GMP-140. Thus, GMP-140 expressed by activated endothelium might promote rapid neutrophil targeting to sites of acute inflammation.     

Purified EGF receptor-kinase interacts specifically with antibodies to Rous sarcoma virus transforming protein

Transformation by several RNA tumour viruses seems to be mediated by virally coded protein kinases which specifically phosphorylate tyrosine. A tyrosine-specific protein kinase also seems to be involved in the mitogenic action of epidermal growth factor (EGF). This EGF-stimulated kinase activity is closely associated with the EGF receptor, with which it copurifies during EGF-affinity chromatography. Because both the virus- and EGF-stimulated tyrosine kinases may be involved in stimulation of cell growth, and because the viral kinases may be antigenically related to normal cell proteins, we examined the interaction of antibodies to viral tyrosine kinases with the affinity-purified EGF receptor-kinase preparation. We report here that the receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60src of Rous sarcoma virus. However, none of these antibodies, including those which cross-react with the normal cellular homologue of pp60src (pp60sarc), precipitate the receptor-kinase. These results suggest that the EGF receptor-kinase is related to, but probably not identical with, pp60sarc.