Biomedical subjects
Publications and source records attributed to K Ohashi.
Four new resin-glycosides named merremosides f (6), g (7), h1 (8), and h2 (9) were isolated from the tuber of Merremia mammosa (Lour.) Hallier f. (Convolvulaceae), an Indonesian medicinal plant. Their chemical structures have been elucidated on the bases of their chemical and physicochemical properties.
Serological survey of bovine immunodeficiency-like virus (BIV) infection was performed in cattle of 3 different farms in Hokkaido, where a relatively high seroprevalence was recorded for bovine leukemia virus (BLV). About a half of 120 cattle tested were seropositive for BLV, while 7.5% of the cattle were seropositive for BIV. Though increased numbers of leukocytes were frequently observed in BLV-seropositive cows, no such changes were observed in BIV-positive but BLV-negative cows. No correlation was demonstrated between BIV- and BLV-seroprevalence of the cattle.
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The efficacy of liposomal FK506 was compared between a canine liver transplantation model and a canine kidney transplantation model. The present study revealed that liposomal FK506 increased immunosuppressive efficacy of FK506 in liver transplantation but decreased in kidney transplantation. Because liposomal FK506 increased FK506 levels in the liver and spleen, and decreased FK506 levels in the kidney, it was suggested that enhanced immunosuppressive efficacy in liver transplantation should be attributed to the local immunosuppressive effects in the hepatic allograft rather than effective suppression of splenocyte activity.
A total of 548 patients had an non-decompression surgery for esophagogastric varices in our hospital, from September 1979 to August 1995. Type of operation includes 402 cases of esophageal transection with paraesophagogastric devascularization (via thoracic approach). 40 cases via abdominal approach, and 106 cases of Hassab procedure. The origin was cirrhosis in 454 patients, IPH in 46, extrahepatic portal occlusion in 29, primary biliary cirrhosis in 6, Budd-Chiari syndrome in 4 and others in 9. Operative mortality rate was less than 1% in child A group, although overall mortality rate including child B, C was 5.0%. By thoracic approach, residual esophageal varices were observed only in 2.5%. Postoperative variceal recurrence were appeared high in cases with hepatocellular carcinoma (HCC). Cumulative recurrence rates at 15 years after surgery were 20.2%, unless HCC had occurred. Cumulative survival rates at 10, 15 years were 52.1%, 45.6% respectively in liver cirrhosis without HCC. Present study confirmed that our operation is effective in controlling esophagogastric varices in long term of periods. Esophagogastric varices of IPH, EHO, and liver cirrhosis of Child A group should be treated by non-decompression surgery.
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Experimental bovine immunodeficiency-like virus (BIV)-infection and mixed infection of BIV and bovine leukemia virus (BLV) were performed on sheep. BIV proviral DNA and anti-BIV antibodies were persistently detected in all BIV-inoculated sheep. A slight increase in lymphocyte counts was observed in BIV-infected sheep, but the percentages of CD4+ and CD8+ cells in sheep peripheral blood mononuclear cells (PBMCs) were not significantly changed. A transient decrease in lymphocyte blastogenic response to concanavalin. A was observed in two of three BIV-infected sheep at 3-6 months after inoculation. From 6 months after BLV-inoculation to sheep which were previously infected with BIV, the numbers of lymphocytes expressing a tumor-associated antigen (TAA) of bovine leukosis were increased compared to those of a sheep inoculated with BLV alone. The BLV titers in PBMCs and the antibody titers against BLV from sheep infected with both BIV and BLV were higher than those of a sheep inoculated with BLV alone.
We previously isolated human cDNA coding for LIMK1 (LIM motif-containing protein kinase-1), a putative protein kinase containing two LIM motifs at the N terminus and an unusual protein kinase domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LIMK1 and 50% overall amino acid identity with LIMK1. The protein kinase domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp-Leu-Asn-Ser-His-Asn in subdomain VIB and a highly basic insert between subdomains VII and VIII. Expression patterns of LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myelin basic protein and histone in in vitro kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The "native" LIMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of "recombinant" LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated protein (pp140) was co-immunoprecipitated with native LIMK1 form A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.
We have isolated cDNA clones encoding the rat and human forms of a novel protein kinase, termed TESK1 (testis-specific protein kinase 1). Sequence analysis indicates that rat TESK1 contains 628 amino acid residues, composed of an N-terminal protein kinase consensus sequence followed by a C-terminal proline-rich region. Human TESK1 contains 626 amino acids, sharing 92% amino acid identity with its rat counterpart. The protein kinase domain of TESK1 is structurally similar to those of LIMK (LIM motif-containing protein kinase)-1 and LIMK2, with 49-50% sequence identity. Phylogenetic analysis of the protein kinase domains revealed that TESK1 is most closely related to a LIMK subfamily. Chromosomal localization of human TESK1 gene was assigned to 9p13. Anti-TESK1 antibody raised against the C-terminal peptide of TESK1 recognized two polypeptides of 68 and 80 kDa in cell lysates of COS cells transfected with human TESK1 cDNA expression plasmid. TESK1 protein expressed in COS cells exhibited serine/threonine kinase activity, when myelin basic protein was used as a substrate. Northern blot analysis revealed that TESK1 mRNA was specifically expressed in rat and mouse testicular germ cells. The TESK1 mRNA in the testis was detectable only after the 18th day of postnatal development of mice and was mainly expressed in the round spermatids. These observations suggest that TESK1 has a specific function in spermatogenesis.
The chromosomal translocation involving 3q27 is often detected in human B-cell lymphomas, especially diffuse lymphomas with a large-cell component. The BCL6 gene has been isolated from the chromosomal breakpoint in these lymphomas. Here we cloned the murine BCL6 (mBCL6) cDNA from the muscle cDNA library using the human BCL6 (hBCL6) cDNA as a probe. The predicted amino acid sequence was 95% identical to that of hBCL6. It contains six repeats of the Krüppel-like zinc-finger motif that are completely identical to those of hBCL6, indicating that the BCL6 gene is well conserved between humans and mice. Expression of the mBCL6 gene was ubiquitously detected in adult mouse tissues including lymphatic organs. Furthermore, it was induced in lymphocytes activated with phorbol ester and Ca2+ ionophore within 30 min after stimulation. This induction was not inhibited by treatment of the cells with a protein synthesis inhibitor, cycloheximide. These results suggest that BCL6 plays a role in activated lymphocytes as an immediate early gene.
In order to investigate the effects of Ca2+, Ba2+ and Sr2+ on vascular endothelial nitric oxide (NO) production, contractile and relaxant responses of porcine depolarized coronary arteries to these divalent cations were compared. In the presence of diltiazem, Ba2+ induced NO-dependent relaxation, Sr2+ did slightly and Ca2+ did not; however all three cations increased cGMP levels in endothelium-intact arteries to similar extents. In the absence of diltiazem, these cations evoked contractions: the EC50 of Ca2+ for endothelium-denuded arteries was lower than those of Ba2+ and Sr2+. The IC50 of diltiazem for arteries precontracted with Ca2+ was higher than for arteries precontracted with Ba2+ and Sr2+. These results suggest that Ba2+ and Sr2+, as well as Ca2+, activate coronary arterial NO production, and also that the different responses of coronary arteries to these divalent cations can be explained, in part, by the different sensitivities of the smooth muscle to these cations and by the different potencies of diltiazem to inhibit the contractions the cations induced.
Sky (also called Rse, Brt, and Tyro3) is a member of a subfamily of related receptor tyrosine kinases, including Axl/Ufo/Ark and c-Eyk/Mer. We obtained evidence that Gas6 (the product of growth arrest-specific gene 6) is a ligand of the Sky receptor tyrosine kinase. Gas6, but not protein S (an anticoagulant protein structurally similar to Gas6), specifically bound to the soluble form of Sky (Sky-Fc), composed of the extracellular domain of Sky fused to the Fc domain of human immunoglobulin G1. The native and recombinant Gas6, but not protein S, stimulated tyrosine phosphorylation of Sky ectopically expressed in Chinese hamster ovary cells. Stimulation of Sky in response to Gas6 was inhibited by Sky-Fc. The half-maximal concentration of Gas6 that stimulated Sky was about 1 nM. Thus, Gas6 as a ligand for Sky specifically binds to and stimulates Sky receptor tyrosine kinase.
The BCL-6 gene encodes a zinc finger-transcription factor and frequently is rearranged in diffuse large cell lymphoma. We have reported here the isolation of the 5'-flanking region of the human BCL-6 gene. Nucleotides sequence of 1.5 Kb promoter region contains TATA box but no CAAT box and a number of potential regulatory elements including CACCC, E-box, and GATA-1 sites. Studies of the promoter activity showed the first 657 nucleotides of the 5'-flanking region significantly drove transcription of the luciferase reporter gene in human Burkitt's lymphoma cell line.
We previously isolated human cDNA encoding LIM-kinase (LIMK), a putative protein kinase which contains two repeats of the LIM motif at the N-terminus and a protein kinase consensus sequence at the C-terminus. Using as a probe a cDNA fragment of human LIMK, we isolated from a rat brain cDNA library cDNA clones encoding two distinct protein kinases (termed LIMK-1 and LIMK-2) related to human LIMK. LIMK-1 shares with human LIMK 95% of the total 647 amino acids and is probably a rat equivalent of human LIMK. LIMK-2 has an overall sequence and a domain structure similar to that of human LIMK and rat LIMK-1, but overall identity is 50-51% at the amino acid level. Like human LIMK, the protein kinase domains of rat LIMK-1 and -2 contain a characteristic sequence DLNSHN in subdomain VIB and a highly basic insert between subdomain VII and VIII. LIMK-1 and -2 are therefore closely related but distinct members of a novel LIM-containing protein kinase subfamily. Several forms of LIMK-2 transcripts encoding proteins that are N-terminally modified and/or C-terminally truncated are generated by alternative splicing or alternative initiation. Northern blot analysis revealed the expression of LIMK-1 mRNA predominantly in the brain and the expression of LIMK-2 mRNA in various tissues in the rat. Antibody raised against LIMK-1 specifically immunoprecipitated and identified in Rat2 fibroblast cells a 72 kDa protein, which has no detectable autophosphorylating activity but is capable of phosphorylating serine and threonine residues of myelin basic protein, by in vitro kinase reaction. As the LIMK family kinases have unique structural features, they are likely to have specific functions in previously uncharacterized signaling pathways.
We previously demonstrated that influenza virus infection induces apoptosis in culture cells. Here, we examined the activation of the Fas antigen gene that encodes an apoptosis-mediating membrane protein in the virus-infected cells. The virus elicited a transient but marked increase in Fas antigen mRNA 3 to 4 hr after infection, followed by the expression of the antigen on the cell surface. Poly(I)-poly(C), a synthetic double-stranded RNA, similarly activated Fas antigen gene expression, and poly(I)-poly(C)-treated cells are highly susceptible to the cell killing effect of IgM isotype of anti-Fas monoclonal antibody. On the other hand, the IgG isotype of anti-Fas monoclonal antibody, which has an inhibitory effect on Fas Ag-mediated cell death, suppressed the virus-induced cell death. Prior exposure of the cells to anti-interferon-beta antibody decreased the degree of cell death as well as the amount of Fas mRNA. The autophosphorylation activity of double-stranded RNA-activated protein kinase was also decreased in the antibody-treated cells. Moreover, a protein kinase inhibitor, 2-aminopurine, blocked the Fas Ag gene activation by poly(I)-poly(C). These results suggested that the activation of Fas Ag gene in the early phase of infection is an important event for apoptosis, and that it is regulated by the double-stranded RNA/interferon system involving protein phosphorylation.
"Sky" is a putative receptor tyrosine kinase predominantly expressed in the brain. Sky, like Axl/Ufo/Ark and c-Eyk, has an extracellular domain composed of two immunoglobulin-like domains and two fibronectin type III domains. Immunoblot analysis using an antibody raised against a C-terminal peptide of Sky identified a 98-kDa Sky protein in COS cells transfected with sky cDNA (COS/sky cells). A 98-kDa protein in the immunoprecipitates with anti-Sky antibody was autophosphorylated on tyrosine, by in vitro kinase reaction. When the lysates of COS/sky cells were immunoprecipitated with anti-Sky antibody and immunoblotted with an anti-phosphotyrosine antibody, a 60-kDa phosphotyrosine-containing protein, in addition to the tyrosine-phosphorylated Sky, was detected. Using the anti-Src antibody, which is reactive to Src, Fyn and Yes, we obtained evidence for an association between the Src family tyrosine kinase and the tyrosine-phosphorylated Sky receptor. These results suggest that the Src family kinase may play an important role in signal transduction of the Sky receptor.
We investigated the effects of intracerebroventricular injection of substance P (SP) on the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance in the mouse. SP (0.001-3 micrograms) alone did not influence either spontaneous alternation performance or total arm entries. Scopolamine (1 mg/kg) impaired spontaneous alternation performance accompanied by an increment in total arm entries. In contrast, SP (0.01-1 micrograms) significantly improved the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance without influencing the scopolamine (1 mg/kg)-induced increase in total arm entries. The effects of SP (0.1 micrograms) on the scopolamine (1 mg/kg)-induced impairment of spontaneous alternation performance were almost completely reversed by pretreatment with WIN 62577 (1 mg/kg), a tachykinin NK-1 receptor antagonist. These results suggest that SP improves the scopolamine-induced impairment of spontaneous alternation performance through the mediation of tachykinin NK-1 receptors.
BACKGROUND: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), we investigated the effects of these two cytokines on death of luteal cells in vitro. METHODS: Mouse luteal cells were cultured in serum-free medium with TNF-alpha at 0, 500, 1,000, 3,000, or 5,000 U/ml in the presence or absence of IFN-gamma at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3' end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. RESULTS: On day 3 of culture, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500-5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-gamma (1,000 U/ml) and TNF-alpha (5,000 U/ml) did. On day 6, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-alpha alone at 5,000 U/ml did, and combinations of IFN-gamma and TNF-alpha at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-alpha. On days 3-6 of culture, combinations of IFN-gamma and TNF-alpha that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. CONCLUSIONS: The presence of IFN-gamma modulates the ability of TNF-alpha to induce a reduction in the number of viable cells, although TNF-alpha alone at high concentrations can induce a reduction in the number of viable cells.