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.     

Michael Addition Polymerizations of Trifunctional Amines with Divinyl Sulfone

Summary: The mechanisms of the Michael addition polymerization of N‐aminoethyl piperazine (AEPZ) with divinyl sulfone (DVS) were clarified based on the reactivity sequence of three different amines in AEPZ: 2° amine in piperazine ring > 1° amine ≫ 2° amine formed in situ. When the feed molar ratio of DVS to AEPZ was 1:1, the polymerization of AB intermediate formed proceeded, and the linear poly(sulfone amine) containing secondary and tertiary amines in the backbones were produced. The linear structure of the product was confirmed by NMR spectra, and the molecular weights, molecular weight distribution, and properties of poly(sulfone amine)s were characterized by GPC, DSC, and TGA.

Polymerization of linear poly(DVS‐AEPZ).     

Multiplicity of allergens in peanuts

Crude peanut protein fractions from raw and roasted peanuts were examined in the RAST with 10 sera from patients showing clinical peanut sensitivity. The radioactive uptake results, which were generally high, did not reveal any distinguishable pattern. Two commercially available peanut proteins, peanut lectin and phospholipase D, gave poor RAST responses. Three purified peanut proteins, α-arachin, conarachin I, and concanavalin A-reactive glycoprotein, all gave significant RAST results that were generally lower than those obtained with the crude extracts. The extent of RAST inhibition obtained with these materials was inversely related to their abundance in the total peanut protein. Crossed immunoelectrophoresis with extracts from raw and roasted peanut indicated the presence of 22 and 10 anodically migrating antigens, respectively. Sixteen IgE binding antigens were revealed for raw peanut and seven for roasted peanut after incubation with a mixed serum from the 10 patients in crossed radioimmunoelectrophoresis (CRIE) using ¹²⁵I-labeled anti-IgE. CRIE plates treated with individual serum samples showed that all the patients had specific IgE for the major antigen peak, which has been tentatively identified as α-arachin. This major storage protein of peanut, which is known to be particularly heat resistant, may be of greater clinical significance than its apparently low RAST activity would seem to indicate.     

改良琼脂糖凝胶电泳法测定血清LDH同工酶的应用

目的 寻求更加准确、简便、快速、实用的乳酸脱氢酶同工酶（LDH）测定方法。方法 据LDH同工酶等电点不同,选择琼脂糖胶作支持物,对以往LDH同工酶测定方法进行改进,将五型LDH同工酶进行分离并定量测定。结果 改进后的LDH同工酶测定方法稳定,区带清晰,重复性好,结果可靠,试剂可长期保存。结论 改良琼脂糖凝胶电泳法测定血清LDH同工酶应用前景良好。     

硅凝胶型乳房假体凝胶外渗的实验研究

目的 :研究硅凝胶型乳房假体植入大鼠体内后凝胶外渗对机体的影响。方法 :取 40只健康SD雌性大鼠分成A、B、C、D 4组 ,A、B、C 3组分别植入实验用完整的硅凝胶型假体、刺破的硅凝胶型假体及生理盐水充注型假体于大鼠背侧皮下 ,D组为空白对照。术前测出血液中有机硅的浓度。 6个月后取出假体观察 ,进行假体包膜组织学检查及大鼠血液中术后有机硅的浓度测定。结果 :A、B、C 3组纤维包膜组织学特征无明显差异 ,B组纤维包膜中光镜下发现外渗的凝胶颗粒。术后与术前 4组大鼠间血液中有机硅的浓度比较 ,差异均无显著性。结论 :完整的硅凝胶假体、人为刺破外壳的硅凝胶假体与生理盐水充注型假体的组织学反应无明显差异 ,外渗的凝胶局限在纤维包膜的内层 ,也不会随血液或淋巴到机体其他部位。     

前列腺雄激素受体亚型的表达及在前列腺内外带的分布

研究雄激素受体(AR)亚型在前列腺组织中的表达及内外带分布。方法采用聚丙烯酰胺凝胶等电聚焦电泳(IEF)技术,检测了前列腺组织标本20例。结果前列腺组织有3种AR亚型,等电点(PI)分别在6.5、6.0和5.3,3种亚型均表达13例,占65％,所有标本均表达PI6.5亚型;前列腺内带和外带均有3种亚型表达,个体间存在差异;定量分析表明,前列腺内外带AR含量,外带高于内带,但无统计学意义(P>0.05),PI6.0亚型浓度在外带明显高于内带(P<0.05)。结论前列腺AR存在亚型并且具有带性分布特征。