括搂素的纯化及其免疫毒素的制备

利用凝胶过滤及离子交换层析从Ｔｒｉｃｈｏｓａｎｔｈｅｓ ｋｉｒｉｌｏｗｉｉ Ｍａｘｉｍｏｗｉｃｚ种子中分离出一种核糖体失活蛋白,称之为括楼素。这种毒素在无细胞体系中对蛋白质生物合成具有明显的抑制活性而对完整细胞的毒性很低。与单克隆抗体偶联后,括搂素对靶细胞显示了明显的特异性细胞毒作用。     

广西植物油脂的研究 Ⅲ.大瑶山九种植物种子的油脂成分

<正> 一、材料 分析样品来源是区综合考察队植物资源组采集。共分析5个科9个种的植物种子:五味子科的冷饭团(Kadsura coccinea)风藤、(K.heteroclita)樟科的广东山胡椒(Lindera kwangtungensis)锈叶新木姜(Neolitsea cambodiana)、葫芦科的大苞括楼(Trichosanthes rosthornii.)、毛括楼(T.mushaensis)、双边括楼(T.uniflora)、蔷薇科的尖嘴林檎(Malus melliana)、省沽油科的大果山香圆(Turpinia pomifera)。     

酵母中细胞自噬的研究进展

细胞自噬是真核生物中高度保守的一类亚细胞降解途径。它通过降解细胞内组分维持细胞内生理平衡并帮助细胞度过逆境。细胞自噬在生物体生长发育、免疫防御、细胞程序性死亡、肿瘤抑制、预防神经退行性病变等方面有非常重要的作用。酵母是目前细胞自噬研究最充分的模式生物,其中分子机制及其他方面的研究成果对整个真核生物细胞自噬的研究有很关键的作用。本综述旨在对近年来酵母中关于细胞自噬的研究进展和常用检测手段作一概括总结。     

DNA疫苗接种的安全性

DNA疫苗的安全性问题主要有三个方面：转入体内的DNA有可能整合到宿主细胞基因组上,使宿主细胞抑癌基因失活或癌基因活化,使宿主细胞转化成癌细胞;外源抗原持续表达产生的不良后果;质粒DNA诱导的自身免疫反应。本综合近年来有关献对DNA疫苗安全性的研究作一概括性介绍。     

肌源干细胞研究进展

目前已证实肌肉是具有多向分化潜能的成体干细胞的一个储存库。研究者认为骨骼肌中至少有两种干细胞：肌卫星细胞（muscle satellite cells）和肌源干细胞（muscle-derived stem cells, MDSCs）,并且使用几种方法从肌肉中分离获得不同类群的MDSCs。研究发现分离这些细胞的方法影响干细胞的特征。本文对MDSCs的行为、生物学特征、分离、分化及其在治疗组织器官修复和再生中应用的可能性等作一概括介绍。     

砂仁种子的解剖学和组织化学研究

砂仁种子包括假种皮、种皮、外胚乳、内胚乳与胚。假种皮由内表皮、外表皮及其间的６－９层薄壁细胞组成。种皮分为外种皮、中种皮与内种皮。外种皮由１层表皮细胞构成,其壁增厚并略木质化。中种皮包括各含１层细胞的下层皮和半透明细胞层与含３－５层细胞的色素层;下皮层与色素层细胞均含有红综色素,后者的壁呈网状增厚。内种皮由１层内切向壁与径向壁非常增厚的石细胞构成。种皮表面具有许多疣状突起,它们是体积较小的表皮细胞     

基于细胞的噬菌体抗体库筛选技术

肿瘤细胞表面抗原的表达量低,免疫原性弱,因抗原构象改变的原因,用传统的固定化抗原方法很难获得有价值的噬菌体抗体。这类抗原大多具有复杂的结构,且其转运、定位与细胞类型及所处的微环境有关。在基于细胞的筛选中,靶抗原以天然状态存在,不需纯化,甚至可以对未知抗原进行筛选,因此广泛用于对内化抗体和血管内皮抗原表位抗体的筛选。基于细胞的抗体筛选中的主要问题是因非特异结合造成的选择背景过高,迄今,已有许多旨在改善选择灵敏度和特异性的研究开展起来。随高通量流式细胞术、组合化学及蛋白质组学研究技术在细胞筛选中的运用,必将使其日趋实用和成熟。我们拟以细胞和组织筛选的技术做一概括,为了解基于细胞的筛选和优化设计提供参考。     

半野生糜鹿集群行为的初步研究

糜鹿在半野生状态下喜集群括动,不同月份集群的大小以及各种组群出现的频率均有所变化。半野生糜鹿有5种组群类型：雄鹿群、母仔群、混合群、雄仔群和仔鹿群。其中混合群出现频率最高,集群最大,是其主要的组群类型,雄鹿群最为稳定,母仔群中采食组群所占比例和组群大小都大于非采食括动的组群,雄仔群和仔鹿群出现频次较少。麋鹿发情期与非发情的集群有明显差异,不同生境内的集群也有所不同。     

干扰素与白细胞介素之间的相互关系

<正> 干扰素与各种白介素(IL-1～6)是由免疫活性细胞诱生的细胞因子(CytoRines),它们作为细胞间相互作用的内源性信号,可以继发地诱导产生其他细胞因子和引起免疫活性细胞间的相互作用(包括正相和负相作用),并对局部和整体的免疫应答都起着重要的调节作用。本文就干扰素与白介素1～6的产生及免疫功能的相互关系综述如下,试图对这些重要的细胞因子的性质、作用及相互关系的最新研究进展有一概括了解。     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion-exchange and gel-filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27,000 with a strong ribosome-inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N-terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3'-dithiobispropionimidate cross-linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A-chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A-chain immunotoxins for in vivo applications.     

Selective low level of protein kinase C isozyme in a tumor promoter-dependent mouse leukemia cell line

It was found that the activity of protein kinase C in the tumor promoter-dependent cell line (A65T) was significantly lower than that in the independent cell line (A65IND) which was mutated from the dependent cell line. On the other hand, there was no significant difference between these cell lines with regard to cAMP-dependent protein kinase activity. It was found that the maximal binding capacity of [20-3H]phorbol-12,13-dibutyrate of the tumor promoter-dependent cells is lower than that of the independent cells with similar affinities of the two cell lines. Moreover, we found that the level of immunoreactive antigen with monoclonal antibody for type III of protein kinase C in A65T cells was significantly lower than that in the A65IND cells. Thus, this selective lower level of type III of protein kinase C in A65T cells, as compared with A65IND cells means that this difference may be linked to its tumor promoter-dependent cell proliferation.     

The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.     

栝楼籽核糖体失活蛋白的纯化、性质及晶体生长研究

栝楼（Ｔｒｉｃｈｏｓａｎｔｈｅｓｋｉｒｉｌｏｗｉ）籽经粉碎抽提、硫酸铵沉淀、阳离子交换及凝胶过滤柱层析等步骤,得到一种单链核糖体失活蛋白－Ｔｒｉｃｈｏｋｉｒｉｎ（ＴＣＫ）．ＳＤＳ－ＰＡＧＥ和ＩＥＦ显示为单一条带,其分子量为２９ｋＤ,ｐＩ≥９．３,含糖量约为１．７５％．该蛋白对兔网织红细胞裂解液系统的蛋白质合成具较强的抑制活性,ＩＣ５０为６．７×１０－１０ｍｏｌ／Ｌ．改进了纯化方法,提高了产率,并培养出晶体．     

Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.     

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

肿瘤及胚胎组织特异表达蛋白CEP65相互作用蛋白的筛选与鉴定

CEP65蛋白(cancer and embryo expression protein 65)为肿瘤单克隆抗体3H11所识别的抗原分子, RT-PCR及RNA印迹检测表明, CEP65蛋白表达于肿瘤及胚胎组织.为了研究CEP65在肿瘤发生中的作用机制,以CEP65为诱饵蛋白,通过酵母双杂交体系筛选人胚胎组织cDNA表达文库. 从5.2×10⁶个克隆中筛选得到14个阳性克隆,并在酵母体系中通过不同方法得到验证.在此基础上,选取在双杂交中作用明显的51号克隆,与CEP65作GST pull-down实验, 结果证明, CEP65与51号阳性克隆蛋白确能相互作用. 生物信息学分析发现, CEP65与51号阳性克隆蛋白相互作用, 可能通过WW结构域或者蛋白激酶C对细胞的增殖与分化起到调节作用.