Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further characterization of the vesicular stomatitis virus temperature-sensitive O45 mutant: intracellular conversion of the glycoprotein to a soluble form.

Reexamination of the viral products of tsO45, a glycoprotein mutant of vesicular stomatitis virus, showed that at 39 degrees C there was a conversion of the glycoprotein (G) to a truncated, soluble form, Gs, which subsequently appeared in the extracellular medium. The half-life for this intracellular conversion and extracellular appearance was about 2 h at 39 degrees C. Gs was precipitated by a monoclonal antibody to the ektodomain but not by an antipeptide serum made against the first 15 amino acids at the carboxy terminus of G. Gs was also resistant to endoglycosidase H digestion. On the basis of pulse-chase experiments, the generation of Gs most probably occurred in the rough endoplasmic reticulum. This additional phenotype of the tsO45 mutant provides another approach for studying the generation and subsequent transport of a secreted protein in fibroblast cells.     

B淋巴细胞在多向造血祖细胞生长中的地位

小鼠骨髓细胞在体外培养中,加入用流式自由电泳法分离所得的高纯度正常B淋巴细胞,可使多向祖细胞(CFU-mix)集落增加至5倍;加入小鼠B淋巴瘤细胞株的条件培基(M_(12.4.1)-CM)时,CFU-mix数也可增加至4倍。单集落形态学分析结果表明M_(12.4.1)-CM可加强CFU-GEMm及p-BFU-E等早期造血祖细胞的增殖与分化。小鼠高纯度B细胞样品在体外培养中加入1000 rad照射的骨髓细胞可出现CFU-mix集落,如果再加入适量的小鼠肺条件培基,则CFU-mix数量比对照大15倍,其集落性质为CFU-GEMm,GMm及p-BFU-E。在此培养中加不同稀释度抗小鼠IgM血清,结果CFU-mix的产率与抗IgM血清的浓度成直线反比关系,当加入1:10抗小鼠IgM血清时,CFU-mix为0。作者假设在一定培养条件下,IgM阳性的部分B细胞可返祖转化为CFU-mix。     

Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization

The inner acrosomal membrane (IAM) develops during the spermatid stage of differentiation as that portion of the Golgi-derived acrosome granule that tightly associates with the condensing sperm nucleus. In some mammalian species, an electron-dense proteinaceous material accumulates between the IAM and the nuclear envelope, collectively comprising the "perforatorium." Evidence, including its partial purification and its structural resistance to detergents and sonication, suggests that the IAM is an unusually resiliant membrane. Dense paracrystalline arrays of intramembranous particles, a lack of lectin-mediated receptor modulation, and its lack of participation in sperm-egg fusion suggest that the IAM lacks the same degree of fluidity as the egg surface plasmalemma. Observations using monoclonal antibodies, however, suggest that some specific antigenic modulations may be possible within the IAM. Its structural rigidity is of obvious mechanical value during sperm penetration through the zone pellucida. An additional role as a scaffold for putative zona lysin material remains controversial. Biochemical evidence suggests that acrosin, for example, is not entirely soluble and that some remains sperm-associated, depending on the conditions of acrosome disruption. Nevertheless, morphological studies do not agree on acrosin's specific localization to the IAM. Currently there is only very limited information concerning the localization of the other acrosomal enzymes to the IAM. Another possible role for the IAM in some species may be in recognizing the zona pellucida. Evidence for this derives from the observation that fucoidin, a fucose heteropolysaccharide, inhibits guinea pig sperm-zona binding, and bound fucoidin can be localized to the IAM and equatorial regions of the living acrosome-reacted spermatozoa. Finally, the IAM may have a role in early recognition/adhesion with the colemma.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

高等植物叶片中的硝酸还原酶活性稳定因子

从小麦、油菜、浮萍、番茄、烟草的叶片中分离得到NR-SF。不同植物材料中NR及NR-SF能起交叉反应;不同NR-SF影响NR酶动力学性质相同;不同NR-SF的凝胶电泳谱带显示蛋白和糖蛋白性质。NR-SF广泛存在于植物细胞中。