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Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 361 records · Page 20Linked to original sources
PubMed1982

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins.

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2-acetamido-2-deoxy-beta-D-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-beta-D-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal alpha-D-mannopyranosyl groups.

Glycopeptides↗
PubMed1982

Studies on endogenous phosphorylation of chicken erythrocyte membranes. Calcium-dependent phosphorylation of specific proteins.

In this work, endogenous phosphorylation of chicken erythrocyte membranes was investigated. The membrane proteins were rapidly phosphorylated endogenously (half maximum time was 30 s) in the presence of millimolar concentration of Mg2+ under physiological conditions. As an exogenous substrate, protamine was phosphorylated most rapidly of those tested, and histone, casein and bovine serum albumin were rather poor substrates. Cyclic nucleotides had no effect on the endogenous membrane phosphorylation. EGTA inhibited the phosphorylation of a membrane protein having an approximate molecular weight of 43000, and this inhibition was reversed by the addition of a stoichiometric amount of Ca2+. Furthermore, trifluoperazine, an inhibitor of calmodulin, was found to have the same effect as that of EGTA. The phosphorylated 43 kDa protein could be extracted from the membranes under high salt conditions, and was precipitated specifically with anti-actin antibody. These results suggest that the phosphorylation of a peripheral membrane protein (which has an approximate molecular weight of 43000) of chicken erythrocytes by membranous protein kinase(s) depends on Ca2+ and possibly on calmodulin.

Adenosine Triphosphate↗
PubMed1982

Separation of acidic oligosaccharides by liquid chromatography: application to analysis of sugar chains of glycoproteins.

An ion-exchange, chromatographic system was developed to separate acidic oligosaccharides. Application of this system for the analysis of sialylated carbohydrate chains of glycoproteins was examined, and it was found that this system affords high resolution of acidic oligosaccharide isomers. (1) 3'-O-Neuraminyllactose, 6'-O-neuraminyllactose, and N-acetyl-6'-O-neuraminyllactosamine could be separated from bovine colostrum. (2) Subjection, to this system, of the oligosaccharide fraction released from bovine submaxillary mucin by treatment with alkaline borohydride resulted in good separation of the trisaccharide-alditol GlcNAc-(1 to 3)-[NeuAc-(2 to 6)]-GalNAcol and the disaccharide-alditol NeuAc- or NeuGc-(2 to 6)-GalNAcol. (3) When the serine- or threonine-linked oligosaccharide fraction obtained from human glycophorin A was subjected to this system, two monosialosylated, trisaccharide-alditol isomers, namely, NeuAc-(2 to 3)-Gal(1 to 3)-GalNAcol and Gal-(2 to 3)-[NeuAc-(2 to 6)]-GalNAcol, could be separated from each other. (4) The separation of so-called triantennary and biantennary, complex type of sugar chains of porcine thyroglobulin was achieved with this system.

Animals↗
PubMed1982

The relationship between phospholipid methylation and calcium influx in murine lymphocytes stimulated with native and modified Con A.

Native Con A and two chemical derivatives, divalent dimeric Con A and monovalent dimeric Con A. induced a transient increase of phospholipid methylation, Ca2+ influx, and also increased DNA synthesis in murine lymphocytes. For each of the individual mitogens, the dose-response curves for these three activities were very similar. However, there were major differences between the dose-response curves for Con A and each of its two chemical derivatives. On the other hand, the time course of phospholipid methylation for each lectin reached a maximum at about 10 min after the addition of lectin, and then gradually decreased to control levels. In like manner, Ca2+ influx reached its maximum at approximately 5 min. The lectin-stimulated increase in phospholipid methylation occurred in calcium-free medium, while the inhibitor of phospholipid methylation, 3-deaza-SIBA, also suppressed the increased calcium influx. This suggests that the Ca2+ influx might be regulated by early phospholipid methylation. Further, in the absence of calcium, the methylated phospholipids do not undergo Con A-accelerated breakdown by phospholipase A2. This suggests that the increased influx of calcium is necessary for the activation of phospholipase A2, an enzyme that hydrolyses methylated phospholipids to yield arachidonic acid and lysolecithin. Blocking any of these biochemical steps also blocked subsequent DNA synthesis, suggesting that the pathway may be required for the activation of lymphocytes.

Animals↗
PubMed1982

Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay.

This paper describes the screening studies of 104 commercial crude drugs for mutagenicity by the rec-assay with Bacillus subtilis as well as the reversion assay with Ames strains TA98 and TA100 of Salmonella typhimurium. The rec-assays showed that 13 water extracts and 27 methanol extracts of the crude drugs were positive. The Ames assays with or without metabolic activation showed that 24 water extracts and 16 methanol extracts were mutagenic. In total, mutagenic activities were found in 45 samples among the 104 crude drugs tested.

Bacillus subtilis↗
PubMed1982

Purification and some properties of a cyclic AMP-binding protein from human erythrocyte membranes.

A cyclic AMP-binding protein of human erythrocyte membranes was solubilized with 0.3% Triton X-100 in 10 mM Tris-HCl (pH 7.4) at 4 degrees C, and purified by DEAE-cellulose column chromatography and affinity chromatography on a cyclic AMP derivative-fixed Sepharose 4B column. The purified cyclic AMP-binding protein showed a single band (molecular weight: 49,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the band was specifically labeled with a photoaffinity analogue of cyclic AMP, 8-N3-cyclic [2-3H]AMP. This protein bound 1.6 mol of cyclic AMP per molecule with an association constant of 3.8 x 10(9) M-1 and the optimum pH for binding was 7.4. The protein inhibited the activity of a purified protein kinase from human erythrocyte membranes [Suzuki, K., Terao, T., & Osawa, T. (1981) J. Biochem. 89, 1--11], while cyclic AMP restored the enzymic activity. The amino acid composition of this protein was different from those of cytoplasmic cyclic AMP-binding proteins. These observations indicate that the cyclic AMP-binding protein purified in this work is the regulatory subunit of a membrane bound cyclic AMP-dependent protein kinase.

Amino Acids↗
PubMed1982

Degradation of band-3 glycoprotein in vitro by a protease isolated from human erythrocyte membrane.

The use of soybean-trypsin-inhibitor-Sepharose-4B to purify a protease present in human erythrocyte membranes is described. The fraction bound in the presence of calcium to the affinity absorbent is active on band-3 glycoprotein in a non-ionic detergent solution at neutral pH. Band-3 glycoprotein is degraded into components having the mobilities of the proteins of bands 4.5, 7 and of lower molecular weights. When calcium is omitted from the membrane extract, an inactive form of this enzyme can be purified. By DEAE-cellulose chromatography this inactive form can be converted into the active form, presumably by dissociation of an enzyme-inhibitor complex.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1982

Effect of p-aminobenzoic acid N-xyloside sodium salt (K-247) on metabolism and functions of normal lymphocytes and leukemic cells.

An N-xyloside derivative of p-aminobenzoic acid, K-247, was investigated for the ability to induce changes of Phospholipid metabolism and membrane transport in murine splenic lymphocytes and leukemic cells. K-247 induced an increase of [3H] methyl group incorporation into phospholipid in both normal lymphocytes and leukemic cells (L-1210 and M1 cells). However, K-247 accelerated the turnover of phosphatidylinositol (PI) measured by [32P] incorporation into PI in L-1210 cells and Ml cells but not in normal lymphocytes. 45Ca2+ influx into normal lymphocytes and leukemic cells was also increased by K-247. A methyltransferase inhibitor, 5'-deoxy-5'-S-isobutyl adenosine (SIBA), suppressed both the increase of phospholipid methylation and that of Ca2+ influx. It seemed that Ca2+ transport might be regulated by membrane phospholipid methylation. On the other hand, K-247 was found to suppress [3H] aminoisobutylic acid (AIB) uptake into L-1210 cells and Ml cells. Protein synthesis in L-1210 cells and Ml cells slightly decreased but RNA and DNA syntheses in both normal and leukemic cells were not affected by K-247. These results suggest that K-247 mainly acts on cell membranes, which are more sensitive to K-247 in leukemic cells than in normal lymphocytes. K-247 also induced differentiation of Ml cells into macrophages and granulocytes with phagocytic activity and morphological characteristics. Moreover, K-247 elevated the Con A response of murine thymocytes, most of which were immature T cells and had low reactivity to Con A, and caused a decrease of Thy 1.2 antigen on thymocytes. It seemed that K-247 also affected maturation of thymocytes.

4-Aminobenzoic Acid↗
PubMed1982

Different cytotoxicity of ricin and a concanavalin A-ricin A chain conjugate between Balb/3T3 cells and the transformed cells.

In the previous paper, we reported that ricin and a concanavalin A-ricin A chain conjugate were more toxic to normal cell lines than the transformed variants. In this work, we confirmed this conclusion using other cell lines (KB 3 flat, ST-balb and R4-IudR) which were derived from the Balb/3T3 clone. KN7-8 had a lower number of Con A binding sites than the sensitive parent line (A-31), while the apparent association constant of Con A and surface receptors KN7-8 was slightly higher than that of A-31. On the other hand, both cell lines had almost the same number of ricin binding sites, while the apparent association constant of KN7-8 was lower than that of A-31. These differences in the number of binding sites and the apparent association constant might not be due to the different sensitivity of these cell lines to the toxin, because the amounts of bound 125I-ricin internalized into the two cell lines during incubation at 37 degrees C were essentially the same. Furthermore, no evidence was obtained showing that there was a difference between A-31 and KN7-8 cells in the ability of reduction of the disulfide bond of ricin, degradation of internalized toxin and in the sensitivity of the protein synthesizing machinery.

Animals↗
PubMed1982

Effect of local anesthetics on the inhibition of protein synthesis by ricin.

In the previous paper, 1) we indicated that local anesthetics such as dibucaine and procaine reduced the toxic action of ricin on Balb/3T3 clone A-31 cells, while these drugs did not show such an effect on KN7-8, a transformed variant of clone A-31. To clarify the different action of the local anesthetics on the cytotoxicity of ricin toward the normal and the transformed cells, some of the possibilities were examined. Dibucaine did not show any influence on the rate of uptake of 125I-ricin into the cells. Furthermore, the rate of reduction of the disulfide bond between the A and B chains, and degradation of ricin in the cells were unaffected by dibucaine. However, the inhibition of the poly U-directed protein synthesizing system from A-31 cells by ricin was reduced in the presence of a low concentration of dibucaine or procaine. Therefore, the most probable explanation for the reduced sensitivity to the toxin in normal cells in the presence of the local anesthetics is that these drugs have some influence on the protein synthesizing system of the normal cells and reduce the inhibitory action of the toxin.

Anesthetics, Local↗
PubMed1982

Elevation of colony stimulating factors in mouse serum after injection of PSK, an antitumor polysaccharide.

The level of colony stimulating factors (CSF) in mouse serum was elevated by a single intraperitoneal injection of PSK, a host-mediated antitumor protein-bound polysaccharide obtained from Basidiomycetes. The assay for CSF activity was performed by employing a semisolid methylcellulose culture method using mouse bone marrow cells, and the activity was estimated by an equation well matched with the dose response curve obtained in the CSF assay. A temporary increase of CSF activity within 10 h after the injection of an antitumor polysaccharide, Krestin (PSK) (250-1000 mg/kg) was followed by a fast decline in the activity, but no significant increases were detected in the cases of 62.5 and 125 mg/kg PSK injections. The CSF activity in PSK (500 mg/kg)-treated mouse serum was separated into two active fractions by DEAE ion-exchange chromatography, and both fractions were found to induce colonies comprised of cells with the properties of macrophage in regard to morphology, cytochemistry of non-specific esterase, phagocytic function and expression of Fc receptors on the cell surface. The elevation of the serum CSF level due to administration of so-called host-mediated antitumor agents might be one of the tumor-defense mechanisms in vivo.

Animals↗
PubMed1982

The modulation of murine immune responses by methyl-B12.

We examined the effect of methyl-B12 on the immune system. As a result, it was revealed that methyl-B12 enhanced antibody production in the in vitro system and that this activity was the strongest with methyl-B12 among the homologues of B12 examined, being nearly as strong as that of Levamisole. It was also revealed that methyl-B12 can exert an enhancing activity on the induction of suppressor T cells Con A. These facts suggest that methyl-B12 has a therapeutic effect against spontaneous incidence of diseases in NZA/W mice. These immunomodulative activities of methyl-B12 and their therapeutic significance against immune disorders were discussed.

Animals↗
PubMed1982

A monoclonal antibody that binds preferentially to human suppressor T cells.

A monoclonal antibody termed JM3-3-3A was produced by somatic cell hybridization. The reactivity was assessed by indirect immunofluorescence. JM3-3-3A was reactive with 41% human peripheral blood T cells and 48% non-T cells. Among various human lymphoblastoid cell lines (MOLT 4F, JM, and TALL-1), TALL-l was found not to be reactive with JM3-3-3A. Human peripheral blood T cells fractionated by JM3-3-3A-coated dish panning were submitted to functional studies. JM3-3-3A positive T cells responded to mitogens, concanavalin A and phytohemagglutinin-P much better than JM3-3-3A negative T cells in the presence or absence of adherent cells. JM3-3-3A positive T cells showed suppressor activity and JM3-3-3A negative T cells showed helper activity in pokeweed mitogen-induced Ig production. More than fifty percent of the JM3-3-3A positive T cells were reactive with OKT8 which binds to suppressor/cytotoxic T cells, whereas 18% of JM3-3-3A negative T cells were reactive with OKT8. In addition, immunoprecipitation experiments identified a protein with an approximate molecular weight of 43,000 as a cell surface antigen for JM3-3-3A. Thus, the reactivity of JM3-3-3A showed a wide distribution but human peripheral blood T cells could be dissected functionally by this antibody.

Animals↗
PubMed1982

Induction of autoimmune phenomena in normal mice treated with natural thymocytotoxic autoantibody.

Natural thymocytotoxic autoantibody (NTA) developed spontaneously in New Zealand Black (NZB) mice consists of two autoantibodies in terms of target cell specificity. One of the autoantibodies, NTA-2, is strongly cytotoxic only against desialized lymphocytes, whereas the other one, NTA-1, is cytotoxic against both intact thymocytes and asialolymphocytes. To study the pathogenic role of NTA in murine autoimmunity, DBA/2 mice were injected every other day with affinity-purified NTA (NTA-1, NTA-2). Control mice received normal mice sera (NMS) or saline. After 20 days of treatment, spleen cells from DBA/2 mice treated with NTA-1 or NTA-2 showed a significant increase in the number of anti-ssDNA plaque-forming cells and IgM-producing cells. Sera from NTA-treated mice showed greater DNA binding than sera from control mice did. The levels of proteinuria were moderately increased in NTA-2-treated mice. Con A responsiveness of thymocytes was markedly reduced in NTA-2-treated mice. On the other hand, Con A-activated spleen cells from both control and NTA-treated mice equally suppressed anti-SRBC antibody production in vitro, suggesting that NTA treatment didn't affect the direct precursors of suppressor T cells. Finally, prior absorption of NTA-1 by thymocytes prevented its ability to induce anti-DNA antibodies; however, prior absorption of NTA-2 by thymocytes didn't affect its activity.

Animals↗
PubMed1982

Guinea-pig lymphotoxin-mediated cytotoxicity and activation of endogenous phospholipase A2.

The effect of cations on lysis of target cells (L . P3 cells) by guinea-pig lymphotoxin (GLT) was examined. Depletion of Ca2+ ions from the extracellular environment caused limited cytolysis, whereas addition of Ca2+ led to enhanced lysis. By applying the GLT pulse technique, it was shown that Ca2+ was not required for the initial step of GLT-mediated cytolysis, the step of binding of GLT molecules to target cell membranes. Among other cations tested, La3+ was found to interfere with the cytolytic action. Neither Mg2+ nor Mn2+ was effective. When target cells whose phospholipids were specifically radiolabelled at the C-2 position with [14C]-arachidonic acid were treated with GLT, radioactivity in the phospholipid fraction decreased. On the contrary, that in the free fatty acid, triglyceride and diglyceride fractions increased. This indicates the existence of endogenous phospholipase A2 that can be activated by GLT. Activation of the enzyme was partially inhibited by addition of EGTA with a concomitant decrease of cytolysis. When GLT was added to a crude membrane fraction of the radiolabelled L . P3 cells, liberation of free fatty acids from phospholipids was readily observed, but the increase of triglyceride-associated radioactivity was negligible. In this case, Ca2+ was found to be prerequisite for the GLT-induced activation of the enzyme. The results suggest that GLT-induced cytolysis includes a substantially Ca2+-dependent rate-limiting stage which comes after the binding of GLT to the target cells. Activation of membrane phospholipase A2 may be at least one of the biochemical changes that constitutes the Ca2+-dependent stage, although it remains to be clarified whether the activation of phospholipase A2 is directly responsible for target cell destruction or not.

Animals↗