Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

Cyclin-Dependent Kinase Inhibitor p21 Modulates the DNA Primer-Template Recognition Complex

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPγS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.     

人促红细胞生成素基因组基因的克隆及全序列分析

为了克隆到全长的人促红细胞生成素基因,从而在真核系统中及转基因动物乳腺中进行表达,从一个中国人的血细胞中提取基因组ＤＮＡ,构建了不完全基因组噬菌体文库,文库效价为５×１０４．这种文库的构建方法是用ＢａｍＨⅠ对基因组ＤＮＡ进行完全酶切,回收１０．５ｋｂ左右的酶切片段,插入到λ－噬菌体ＥＭＢＬ３载体中．筛选文库所使用的探针是用ＰＣＲ方法从基因组ＤＮＡ模板中扩增的５５６ｂｐ的ＥＰＯ基因片段．从该文库中筛选到了一个含有完整ＥＰＯ基因的插入片段长度为１２．５ｋｂ的阳性克隆．经全序列分析证实,所克隆的ＥＰＯ基因编码正确,但在５′－侧翼及第一内含子处与国外发表的序列相比较,发现两处核苷酸差异,这两个核苷酸的差异改变了５′－调控区的两个小的开放阅读框——ＯＲＦ１和ＯＲＦ３,其在基因表达调探方面的作用值得进一步探讨．     

反义RNA调节肿瘤细胞O~6-甲基鸟嘌呤-DNA甲基转移酶活性

报道了逆转录病毒载体介导的反义ＲＮＡ对肿瘤细胞Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）活性的调节作用．构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体并用它们转染ＨｅＬａＳ３肿瘤细胞,观察细胞在转染前后ＭＧＭＴｍＲＮＡ水平、ＭＧＭＴ活性及其对ＡＣＮＵ抗药性的变化．发现针对ＭＧＭＴｍＲＮＡ５’端的反义ＲＮＡ能够有效地降低ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并使细胞对ＡＣＮＵ的敏感性提高４．６倍;针对ＭＧＭＴｍＲＮＡ全长的反义ＲＮＡ也能在一定程度上调节细胞的ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并增加细胞对ＡＣＮＵ的敏感性,而针对３’端序列的反义ＲＮＡ对ＭＧＭＴ活性没有调节作用．     

叶绿素荧光动力学参数的意义及讨论

叶绿素荧光动力学技术被称为研究植物光合功能的快速、无损伤探针。但其参数众多，且名称及在参数的生物学意义解释上存在不规范和混乱现象。本文通过对这些问题的讨论旨在引起使用者的注意，并探讨正确使用这些参数的途径     

野生大豆与栽培大豆rDNA ITS1区的研究

采用ＰＣＲ 技术从野生大豆（Ｇｌｙｃｉｎｅ ｓｏｊａ）、半野生大豆（Ｇ．ｇｒａｃｉｌｌｉｓ）、多年生野生大豆（Ｇ．ｔｏｍｅｎｔｅｌｌａ、Ｇ．ｔａｂａｃｉｎａ）和栽培大豆（Ｇ．ｍ ａｘ）的两个品种ＵＮＩＯＮ、文丰７ 中扩增和克隆了ｒＤＮＡ第一转录间隔区（ＩＴＳ１）。其在Ｇ．ｍ ａｘ 基因组中的拷贝数约为２×１０３。序列分析表明Ｇ．ｓｏｊａ、Ｇ．ｇｒａｃｉｌｌｉｓ、Ｇ．ｍ ａｘ 中的Ｇ／Ｃ含量为６１．４０％ ,而Ｇ．ｔａｂａｃｉｎａ和Ｇ．ｔｏｍ ｅｎｔｅｌｌａ的Ｇ／Ｃ含量分别为５８．１１％ 和５９．０１％ ,与绿豆Ｇ／Ｃ含量（５９．８１％ ）相近。Ｇ．ｔａｂａｃｉｎａ的Ｇ／Ｃ含量是已知的植物中ＩＴＳ１ 最低的。最大同源性分析表明,大豆属植物ＩＴＳ１ 的同源程度很高,同其近缘属绿豆的同源性明显高于其它作物。同时分析了栽培、多年生和一年生野生大豆间的亲缘关系。另外还发现在已知的植物ＩＴＳ１ 序列中均含有ＧＡＣＣＣＧＣＧＡＡ 及ＧＣＧＣＣＡＡＧＧＡＡ 两个区段     

九个水稻耐盐突变体的RFLP分析

用６ 个可能与水稻耐盐性有关的ＤＮＡ 探针对来自两个品系的５ 个耐盐突变株系、３ 个耐盐突变体及１ 个弱耐盐突变体进行ＲＦＬＰ分析。结果有８ 个耐盐突变体（株系）检测到ＤＮＡ 水平的变化,６ 个探针检测到具多态性的突变株系（体）的数目分别为ＲＧ４：６ 个;ＲＧ７１１：６ 个;Ｒａｂ１６：５ 个;Ｒａｂ１０Ｅ６：２ 个;Ｒａｂ２１：１ 个;ＳａｌＴ：２ 个;表明ＲＧ４、ＲＧ７１１ 及Ｒａｂ１６三个位点有可能与耐盐性突变相关。多态性图谱中７０．８％ 为２ 个以上的酶切图谱同时显示多态性,说明多数突变是由缺失、插入或重复造成的     

经蛋白A连接于琼脂糖的单克隆抗体亲和层析柱用于抗独特型抗体的纯化

 以Sepharose CL-4B-Pro A吸附胃癌单克隆抗体(McAb)PD4,继之以交联剂二甲基庚二亚胺二盐酸盐(dimethyl pimelimidate dihydrochloride)处理,形成亲和介质(柱Ⅰ)。该亲和介质用于纯化抗PD4独特型抗体(aIdAb)具有明显的优点。与Sepharose CL-4B-PD4(柱Ⅱ)相比,前者的结合容量为后者的1.78～1.94倍。采用柱Ⅰ纯化的aIdAb,当其与McAb PD4的分子比分别为1:1及4:1时可50％或100％地抑制McAb PD4与靶细胞的结合,但采用柱Ⅱ获得的aIdAb,只有当分子比达到2:1及8:1时,才能达到同等的抑制效应。这可能是由于Pro A与McAb PD4的Fc片断结合,使后者的Fab端得以充分暴露,因而有更多的机会与aIdAb结合。     

Upregulation of gamma‐2 laminin‐332 in the mouse ear vesicant wound model

Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin‐332 (formerly LN‐5), a heterodimer formed from α, β, and γ polypeptide chains. Under static conditions, laminin‐332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin γ2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin‐332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real‐time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin‐γ2 in comparison to the other two chains. Protein production of laminin‐γ2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin‐332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin‐γ2. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:172–184, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20275