Bovine virus diarrhea-mucosal disease. II. Isolation and characterization of a cytopathogenic virus and experimental production of the disease.

A disease broke out in calves in the Tokachi district of Hokkaido. It induced pyrexia, respiratory symptoms, diarrhea, bloody feces, leukopenia, and sometimes erosion of the oral mucous membrane and muzzle. Its morbidity rate was 90% and its fatality rate 50%. Bovine virus diarrhea (BVD) virus was isolated from organs of dead calves and blood and feces of affected calves. It exhibited a cytopathic effect on calf kidney cell culture. Antisera against the Nose and the Oregon C24V strains of BVD virus showed an antibody titer of the same order against the homologous virus and the isolated strain. Antiserum against the isolated strain, however, showed much lower antibody titers against the Nose and the Oregon C24V strains than against the homologous virus. When inoculated with the isolated virus, two calves manifested acute symptoms, but recovered at any rate. One of them, however, suffered again from clinical infection and died eventually 37 days after inoculation. It presented pathological changes closely resembling those of the case of spontaneous infection. Virus was recovered from its principal organs, intestinal canal, and lymph nodes of various regions of the body.     

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.     

Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes.

To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells.     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp.

Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglio- and lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.     

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.     

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wishar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI₂-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to Λ¹⁷-6-keto-PGF_1α, a stable metabolite of PGI₃, could not be detected by an incubation study of ¹⁴C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI₂-like substance produced by rat aorta is most likely to be PGI₂. itself and not PGI₃.     

Effect of hypophysectomy and growth hormone administration on somatostatin and gastrin content in the stomach of rats

The effect of hypophysectomy and bovine growth hormone (GH) administration on somatostatin (SRIF) content as well as gastrin content in the rat stomach was investigated. SRIF content was determined by a specific radioimmunoassay. The total SRIF content in the stomach had decreased 4 weeks after hypophysectomy but was restored significantly in those rats which were subjected to bovine GH administration for 7 days after hypophysectomy. Furthermore, in control rats, an increase in SRIF content in the stomach was observed after 7 days of GH administration. Similar changes in total content of gastrin were observed after hypophysectomy and bovine GH administration, although these changes were not significant. These results indicate that GH may influence gastric function through changes in SRIF and gastrin content in the stomach.     

Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.