鼻咽癌转移相关的分泌蛋白质的筛选

目的：筛选鼻咽癌（nasopharyngeal carcinoma, NPC）转移相关的分泌蛋白质，为阐明NPC转移机制以及筛选NPC转移分子标志物提供实验依据。方法：应用二维凝胶电泳（two-dimensional electrophoresis，2-DE）技术分离一对来自同一亲本，具有不同转移潜能的NPC细胞系5-8F和6-10B细胞的分泌蛋白质，图像分析识别差异表达的蛋白质点，基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾-四极杆-串联质谱(ESI-Q-TOF MS/MS)对差异表达的蛋白质点进行鉴定。Western印迹法检测差异分泌蛋白质nm23-H1在两株细胞中的表达水平。结果：建立了5-8F和6-10B分泌蛋白质的2-DE图谱，质谱分析鉴定出14个非冗余的分泌蛋白质，其中Oncoprotein18等蛋白质在高转移潜能NPC细胞系5-8F中的表达水平高于无转移潜能的NPC细胞系，而nm23-H1等蛋白质在5-8F细胞系中的表达水平低于6-10B细胞系。Western印迹分析证实了nm23-H1在5-8F和6-10B细胞分泌蛋白质中的差异表达水平。结论：所鉴定的14个非冗余的差异分泌蛋白质为研究NPC转移机制以及筛选NPC转移分子标志物提供了实验依据。     

Multiple Separations in Microfabricated Channels: From Biological Microenvironments to DNA

A continuous sample introduction and separation scheme is presented as an alternative to the current slab gel and microfabricated chip technologies for biological separations. This new technique involves continuous sample introduction via a conventional small bore capillary coupled to a small dimension rectangular channel. Both free zone and size based separations have been carried out in the rectangular channel. Laser induced fluorescence and electrochemical detection schemes have been employed with this technique. Some of the areas this technology has been used to investigate include monitoring dynamic events from microenvironments, monitoring analytes over long periods of time, and performing DNA separations.     

上海人群面肩肱型肌营养不良症相关位点的D4Z4多态性

目的 对上海人群4p35位点D4Z4重复序列进行研究，分析D4Z4的多态性。方法 191名正常上海人的基因组DNA经EooR I/Bln I双酶水解后，应用脉冲场凝胶电泳及Southem印迹测定其染色体4p35位点的D4Z4片段长度，并对短的D4Z4片段Kpn I酶进行部分酶切以计数其D4Z4串联重复序列数。结果 在191名正常上海人群中，有17人（占8.9％）携带短的D4Z4片段，其长度在22－34kb之间；其中16人携带的短D4Z4片段位于4q35位点，1人携带的短D4Z4片段为4q35→10q26。结论 面肩肱型肌营养不良症的发病虽与4q35位点D4Z4片段的串联重复序列数减少有关，但上海人群中携带4q35位点短的D4Z4片段个体的比例明显高于西方人群，提示其他因素可能也参与面肩肱型肌营养不良症的发病。     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.     

Pulsed-field gel electrophoresis as an epidemiologic tool for enterococci and streptococci

Enterococci (Enterococcus faecium and Enterococcus faecalis) and streptococci such as Streptococcus pyogenes (Group A streptococcus), Streptococcus agalactiae (Group B streptococcus), and Streptococcus pneumoniae are increasing in importance as both hospital-acquired and community pathogens. Emerging resistance and increasing incidence of these organisms has necessitated the analysis of their epidemiologic mechanisms of spread. Pulsed-field gel electrophoresis (PFGE) has emerged as the one of the most widely applicable, reproducible, and stable methods to examine strain identity in bacterial organisms. The procedure used in our laboratory for PFGE typing of whole cell DNA digested with SmaI for enterococci, S. pneumoniae, S. pyogenes, and S. agalacatiae is presented. Issues regarding interpretation are also reviewed and discussed.     

Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.     

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

A novel molecule expressing HLA-DR antigenic determinants

Our previous studies suggested that the polymorphism of HLA-DR antigens (the human equivalent of murine I-E antigens) was a result of structural variation in the small (beta) subunit. In order to more accurately define this polymorphism we have expanded these studies to include HLA-DR antigens isolated with monoclonal cells derived from genotypically HLA-homozygous DRw2, DR2w5, and DRw7 lymphoblastoid cells derived from offspring of consanguineous relationships. Our results indicate the large (alpha) subunits of DRw2 and DRw7 antigens are nearly identical, while their beta subunits show many differences. In contrast, both the alpha and beta subunits of the DRw5 antigen differ strikingly from the respective subunits of the DRw2 and DRw7 antigens. The significance of the variability of the DRw5 alpha subunit is in question at this point. One intriguing possibility is that DRw5 actually represents the human counterpart of the mouse I-A subregion antigen and that the monoclonal antibody is reacting with a determinant which is shared by the human equivalents of murine I-A and I-E antigens.