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Biomedical subjects

H Teraoka

Publications and source records attributed to H Teraoka.

At least 109 records · Page 6Linked to original sources
PubMed1991

Adhesion of immature thymocytes to thymic stromal cells through fibronectin molecules and its significance for the induction of thymocyte differentiation.

Only 10-15% of unseparated thymocytes adhered to culture plates precoated with fibronectin (FN), but 60-70% of the CD4-8- (double-negative) thymocyte population did. This population bound to FN but not to collagen, laminin, or vitronectin. Its binding to FN was inhibited by anti-FN antibody or a mixture of synthetic peptides corresponding to two different sites of FN, termed the V10 sequence and the RGDS (Arg-Gly-Asp-Ser) sequence, which interact, respectively, with the VLA-4 and VLA-5 FN receptors expressed on T-lineage cells. CD4-8- thymocytes also adhered to a monolayer of a thymic stromal cell clone, MRL104.8a, that induces growth-maintenance and differentiation of such thymocytes. The involvement of FN-FN receptor interaction in this adhesion was demonstrated by the following lines of evidence: (i) the MRL104.8a cells expressed FN molecules on their surface and (ii) the adhesion of CD4-8- thymocytes to MRL104.8a monolayers was almost completely inhibited by simultaneous addition of anti-FN antibody and a mixture of peptides (V10 plus RGDS) capable of binding to anti-FN receptors (VLA-4 and -5). Most important, blocking the adhesion of CD4-8- thymocytes to the thymic stromal cell monolayer resulted in potent inhibition of the differentiation of these thymocytes, which was otherwise induced toward the expression of CD4 and/or CD8 molecules. These results indicate that immature CD4-8- thymocytes adhere to thymic stromal cells preferentially through FN-FN receptor interaction and that such adhesion has a critical role in inducing and/or supporting the differentiation of these thymocytes.

Animals↗
PubMed1991

rig encodes ribosomal protein S15. The primary structure of mammalian ribosomal protein S15.

rig, a gene originally isolated from a rat insulinoma cDNA library, codes for a basic 145 amino acid protein [( 1986) Diabetes 35, 1178-1180]. Here we show that the immunoreactivity to a monoclonal antibody against the deduced rig protein and the translation product of rig mRNA comigrated with ribosomal protein S15. The amino acid sequence of ribosomal protein S15 purified from rat liver coincided with that deduced from the nucleotide sequence of rig mRNA, but there were indications that the initiator methionine was removed and the succeeding alanyl residue was monoacetylated. From these results, we conclude that the product of rig is ribosomal protein S15.

Amino Acid Sequence↗
PubMed1991

Glucocorticoid treatment reduces prostacyclin synthesis in response to limited stimuli.

Dexamethasone 21-acetate (DMS 21-A) time- and dose-dependently suppressed bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells. The suppression was more prominent in the presence of pertussis toxin, which by itself could enhance bradykinin-induced prostacyclin synthesis. The DMS 21-A treatment diminished prostacyclin synthesis also in response to vasopressin. In contrast, it did not affect prostacyclin synthesis in response to arachidonic acid or A23187. Melittin-induced prostacyclin synthesis was reduced only at low doses (1-7 x 10(-7) M). The suppression of bradykinin-induced prostacyclin synthesis by DMS 21-A was completely blocked by cycloheximide. DMS 21-A had no effect on the cellular level of lipocortin I protein, but increased the anti-phospholipase A2 activity in EDTA extracts of the cells. These results suggest that the DMS 21-A treatment induces phospholipase A2 inhibitor protein(s) other than lipocortin I and reduces prostacyclin production in response to limited stimuli.

Animals↗
PubMed1991

Expression of c-fos and c-myc in Raji Burkitt's lymphoma cells during the progression of DMSO-induced G1 cells into S phase.

Using flow cytometry, we have recently found that dimethyl sulfoxide (DMSO) reversibly induces G1-arrest in the cell cycle of human lymphoid cell lines such as Raji, Akata, and Molt-4. Here we investigated c-fos and c-myc expression in Raji Burkitt's lymphoma cells at DMSO-induced G1 arrest and after release from DMSO. A small but significant accumulation of c-fos mRNA was observed in G1-arrested Raji cells after treatment with 1.5% DMSO for 96 h. When G1-arrested Raji cells were transferred to DMSO-free medium, a transient increase in c-fos transcripts was detected 30-60 min after the release. The steady state level of c-myc transcripts in G1-arrested Raji cells was found to be one-third that in the log-phase cells. After removal of DMSO, the level of c-myc mRNA was restored and reached a maximum at 4.5-6 h. Immunoblot analysis with a monoclonal antibody against human c-myc protein indicated that c-myc protein in the G1-arrested cells was decreased less than one-tenth that in the log-phase cells. The level of c-myc protein gradually increased after the release from DMSO and reached a maximum at 6-9 h.

Blotting, Northern↗
PubMed1991

cAMP negatively regulates mRNA levels of actin and tropomyosin in rat cultured vascular smooth muscle cells.

The isoform expression patterns of actin and actin-binding proteins have been reported to be good markers for phenotypic modulation of rat vascular smooth muscle cells. In order to elucidate the regulatory mechanism of actin and tropomyosin isoform expression on a molecular basis, we examined the effects of various agents on the isoform expression patterns of actin and tropomyosin at the mRNA level in smooth muscle cells. We found that cAMP-elevating agents induced drastic decreases in the amounts of alpha-smooth muscle type actin and alpha-tropomyosin transcripts, the expression of other actin and tropomyosin isoforms being also repressed, but to a lesser extent. The results of the experiment involving RNA synthesis inhibitors strongly suggest that activation of some mRNA-specific degradation machinery by cAMP might be responsible at least for the rapid disappearance of alpha-smooth muscle type actin and alpha-tropomyosin transcripts in smooth muscle cells.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Two types of DNA ligase I activity in lymphoblastoid cells from patients with Bloom's syndrome.

DNA ligases I and II were separated by hydroxylapatite (HA) column chromatography in cell-free extracts of lymphoblastoid cell lines (LCLs) derived from two unrelated patients with Bloom's syndrome (BS) and two healthy individuals. The specific activity of ligase I from the crude extract was consistently lower in GM3403, a BS LCL from an Ashkenazi Jewish patient, than in normal control LCLs. By contrast, the level of ligase I activity in BSL-2KA, another BS LCL derived from a Japanese patient, was equivalent to those in normal LCLs, although GM3403 and BSL-2KA shared the feature of exceedingly high frequency of spontaneous sister-chromatid exchange. The levels of total ligase activity in crude extracts without the separation into the two forms, however, were approximately two-fold higher for the two BS LCLs than for the normal LCLs. Partial purification by chromatography on a DEAE-cellulose 23 column and a phosphocellulose column did not affect the superiority of the two BS LCLs over the normal LCLs in the specific activity of the total ligases. Nonetheless, subsequent application to an HA column again resulted in much less elevation of the specific activity of ligase I for GM3403 than for BSL-2KA and control LCLs. The levels of ligase II activity, accounting for 4-13% of total ligase activity, were similar among the LCLs examined. Irrespective of the extent of purification, essentially no difference in the heat lability of DNA ligase I was detected among the four LCLs. These findings suggest that there may exist among BS LCLs at least two types of subtle abnormality of DNA ligase I itself and/or a putative substance modulating the enzyme function.

Bloom Syndrome↗
PubMed1991

Ryanodine inhibits caffeine-evoked Ca2+ mobilization and catecholamine secretion from cultured bovine adrenal chromaffin cells.

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.

Adrenal Glands↗
PubMed1991

Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction.

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.

Bacterial Proteins↗
PubMed1991

[Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction].

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.

Bacterial Proteins↗
PubMed1990

Enhancement of bradykinin-induced prostacyclin synthesis in porcine aortic endothelial cells by pertussis toxin. Possible implication of lipocortin I.

Bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells was enhanced by pretreatment of the cells with pertussis toxin or islet-activating protein (IAP) for 5 hr or longer. Although ADP-ribosylation of a protein with a molecular weight of 41-42 kD in the cell membranes was completed by 3 hr after the addition of IAP into the incubation medium, there was good correlation between enhancement of bradykinin-induced prostacyclin synthesis and ADP-ribosylation of the IAP substrate over a wide range of IAP concentrations. Furthermore, even if IAP was removed from the incubation medium at 3 hr, bradykinin-induced prostaglandin synthesis at 24 hr was still potentiated. Cycloheximide and actinomycin D enhanced bradykinin-induced prostacyclin synthesis and apparently blocked the effect of IAP. Since this result suggested the involvement of an inhibitor protein(s) of prostacyclin synthesis in the IAP effect, we studied the effect of IAP on the level of lipocortin I which is known to inhibit phospholipase A2. Western and Northern blot analyses revealed that IAP decreased the amounts of protein and mRNA of lipocortin I. These results suggest that the enhancement of bradykinin-induced prostacyclin synthesis by IAP is associated with a decrease in the level of lipocortin I.

Adenosine Diphosphate Ribose↗
PubMed1990

A simple and sensitive method for determining transcription initiation site: identification of two transcription initiation sites in rat group II phospholipase A2 gene.

We developed a simple and sensitive method for assigning transcriptional initiation sites, and applied it to characterize the transcriptional unit of rat group II phospholipase A2 (PLA2) gene. Our method involves the primer extension reaction followed by detection of its products by hybridization. Using this method, we were able to map two transcriptional initiation sites on the nucleotide sequence of the core promoter region of PLA2 gene with one-base resolution without any difficulties.

Animals↗
PubMed1990

Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.

reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

Amino Acid Sequence↗
PubMed1990

Glucocorticoids suppress group II phospholipase A2 production by blocking mRNA synthesis and post-transcriptional expression.

We investigated the effects of glucocorticoids on group II phospholipase A2 (PLA2) expression in rat cultured smooth muscle cells. Both forskolin-induced and tumor necrosis factor (TNF)-induced PLA2 release responses were almost completely blocked by 10 and 100 nM dexamethasone, respectively, as assayed by protein blotting and PLA2 activity assays. Dexamethasone-mediated inhibition of PLA2 release appeared to be mediated by the glucocorticoid receptor. Dexamethasone at concentrations greater than 10 nM inhibited forskolin-induced elevation of the group II PLA2 mRNA level but not TNF-induced elevation. These data suggest that the mechanism mediating forskolin-induced mRNA accumulation is sensitive to glucocorticoids, but the mechanism mediating the TNF-induced accumulation is not. Inhibition of TNF-induced PLA2 release by glucocorticoids may be explained by the blocking of post-transcriptional synthesis of the group II PLA2.

Animals↗
PubMed1990

The role of lipocortin I in macrophage-mediated immunosuppression in tumor-bearing mice.

The soluble mediators and/or mechanisms involved in immunosuppression in tumor-bearing hosts are not well characterized, although macrophages have long been recognized as major participants. We have investigated the role of lipocortin I, a phospholipid-binding protein, in macrophage-mediated immunosuppression in tumor-bearing mice. Proliferation of splenic lymphocytes in response to the mitogens (PHA, Con A, LPS, and PWM) was severely suppressed in tumor (Sqc-NH-1 carcinoma)-bearing mice. This immunosuppression was associated with a decrease in T and B lymphocytes and an increase in macrophages in these spleens. Mac-2+ macrophages were found only in spleens from tumor-bearing mice. Splenic macrophages from tumor-bearing, but not normal, mice were responsible for this immunosuppression, as revealed by negative and positive selection experiments. The levels of lipocortin I mRNA expression were markedly increased in peripheral blood cells from tumor-bearing mice as compared with those from normal mice. Lipocortin I mRNA was strongly induced in splenic mononuclear cells from tumor-bearing mice. Furthermore, these cells displayed increased expression of lipocortin I protein, as judged by Western blot analysis with polyclonal anti-lipocortin I serum. Some nonimmune organs such as the heart, submaxillary gland, muscle, and bladder also displayed increased levels of lipocortin I mRNA expression in tumor-bearing mice. Mac-2+ macrophages among the splenic mononuclear cells in tumor-bearing mice expressed lipocortin I mRNA, as judged by negative and positive selection experiments. Most of these Mac-2+ macrophages also had Mac-1 and Mac-3 Ag. Lipocortin I protein was increased in the serum of tumor-bearing mice as compared with normal mice. The culture supernatants of splenic cells from tumor-bearing mice suppressed the mitogenic responses of splenic cells from normal mice, and addition of anti-lipocortin I antiserum inhibited this suppression. Furthermore, recombinant mouse lipocortin I suppressed mitogenic responses of splenic cells from normal mice. In summary, Mac-2+ macrophage-derived lipocortin I was largely involved in immunosuppression in tumor-bearing mice.

Animals↗
PubMed1990

Transfection of cDNA with G----T point mutation at the cleavage site of insulin receptors to COS 7 cells.

To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance.

Amino Acid Sequence↗
PubMed1990

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.

1-Methyl-3-isobutylxanthine↗