Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.     

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

牛病毒性腹泻病毒单克隆抗体的研究

牛病毒性腹泻——粘膜病是世界性广泛流行的奶牛和肉牛的传染病。其病原为牛病毒性腹泻病毒(BVDV),属于披膜病毒科的瘟病毒属,它的许多生物学特性至今还不很清楚。本试验建立了12株分泌抗BVDV的单克隆抗体(McAb)杂交瘤细胞株,并结合免疫转移电泳法和放射免疫沉淀法,初步研究了BVDV的多肽。     

Glutathione mixed disulfide inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

Six glutathione-containing inhibitors of the human NADP-linked 15-hydroxyprostaglandin dehydrogenase have been isolated from placental homogenates. Glutathione disulfide is one of these inhibitors. Although the structures of the other five have not been fully elucidated, all are disulfides. Studies with these compounds and with other mixed disulfides have shown that the glutathione mixed disulfides of β-mercaptopyruvate, mercaptoacetate, and β-mercaptolactate are more effective inhibitors of the enzyme than are the glutathione-containing mixed disulfides isolated from placental homogenates. β-Mercaptolactate is particularly noteworthy because of its low K_j (0.13 μM). The results reported here suggest that the activity of the prostaglandin dehydrogenase may be influenced by various glutathione mixed disulfides.     

Effect of platelet-activating factor on airway vascular permeability: possible mechanisms

We studied the effects of the potent inflammatory mediator, platelet-activating factor (PAF), on vascular permeability in airways (and other tissues) of guinea pigs by measuring extravasation of circulating Evans blue dye. PAF caused a dose-dependent increase in vascular permeability. At 1 ng/kg iv, PAF caused an increase in Evans blue extravasation of 220% (P less than 0.05) in the trachea, with the greatest effect at a dose of 100 ng/kg (858%; P less than 0.01). Histamine (150 micrograms/kg iv) caused a 320% increase over base line in the trachea and 200% in main bronchi; this effect was equivalent to that induced by 10 ng/kg PAF in the trachea and 1 ng/kg in main bronchi. The duration of effect of PAF was greatest in main bronchi (less than 10 min). Platelet depletion with a cytotoxic antibody, or the cyclooxygenase inhibitor, indomethacin, or the cyclooxygenase-lipoxygenase inhibitor, BW 7556, did not affect the vascular permeability response to PAF. The PAF-receptor antagonist, BN 52063, inhibited Evans blue extravasation in the airways in a dose-dependent manner, with complete inhibition at 5 mg/kg. Thus PAF-induced airway vascular leakage is mediated by specific receptors but not by products of arachidonic acid metabolism or by platelets. Increased airway microvascular leakage induced by PAF may lead to plasma extravasation and airway edema, factors that may contribute to the airway narrowing and hyperresponsiveness induced by PAF.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231–246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83–90), which is essential for the biosynthetic activities of the embryo.