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Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 325 records · Page 18Linked to original sources
PubMed1985

Purification and characterization of four isolectins of mushroom (Agaricus bisporus).

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.

Agaricales↗
PubMed1985

Translation of human macrophage activating factor (for glucose consumption) mRNA in Xenopus laevis oocytes.

Total messenger RNA was extracted from a human T cell hybridoma, clone H-E4-9, which strongly produced macrophage activating factor for glucose consumption (MAF-G). This messenger RNA gave rise to functional MAF-G when translated in Xenopus laevis oocytes. Isoelectric focusing of culture supernatants of the mRNA-microinjected oocytes and the H-E4-9 cells revealed that the former contained MAF-Gs with isoelectric points of pH 5.0 and 3.0 while the latter contained MAF-Gs with isoelectric points of pH 5.0 and pH 3.3. Sucrose density gradient centrifugation analysis showed that MAF-G mRNA prepared from H-E4-9 cells sedimented at about 11.5 S.

Animals↗
PubMed1985

Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice.

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.

Adenosine↗
PubMed1985

Natural killer (NK) cell activating factor released from murine thymocytes stimulated with an anti-tumor streptococcal preparation, OK-432.

Natural killer (NK) activity of mouse splenocytes was significantly augmented when the splenocytes were incubated for 3 to 4 hr with culture supernatants of mouse thymocytes stimulated by OK-432, an antitumor preparation from the Streptococcus pyogenes SU-strain. Antiviral activity was also detected in the culture supernatants, but IL 2 activity was not. When the culture supernatants of thymocytes stimulated by OK-432 were fractionated on a column of Blue Sepharose CL-6B, NK enhancing activity and antiviral activity were observed in partly overlapping fractions that bound to the column. However, the antiviral activity in the Blue Sepharose-bound fraction was neutralized completely by treatment with anti-IFN (alpha, beta) antiserum, whereas significant NK cell enhancing activity was still observed after treatment with anti-IFN (alpha, beta) antiserum. When the Blue Sepharose-bound fraction was subjected to gel filtration, the NK cell enhancing activity was detected in the 25,000 to 35,000 and 40,000 to 67,000 m.w. regions, but antiviral activity was observed in the over 67,000 m.w. region. These results indicate that a new kind of lymphokine, called natural killer cell activating factor (NKAF), distinct from IFN and IL 2, was found. The NKAF was found to have the following properties: its pI value is between pH 5.5 and 6.5, it binds to concanavalin A- and lentil agglutinin-Sepharose, and it is stable with pH 2-24 hr treatment. In addition, NKAF-producing cells were peanut agglutinin (PNA)-thymocytes when thymocytes were fractionated by the agglutination-sedimentation method with the use of PNA.

Animals↗
PubMed1984

Lectin-like molecules on the murine macrophage cell surface.

Lectin-like molecules on the surface of murine peritoneal exudate macrophages induced by thioglycolate or an anti-tumor streptococcal preparation, OK-432, were investigated and isolated. Furthermore, their sugar-binding specificities and their role in macrophage-mediated tumor cytotoxicity were examined. A neoglycoprotein, D-galactose (Gal)-bovine serum albumin, bound to these murine peritoneal macrophages. This binding of Gal-bovine serum albumin was inhibited by D-galactose, and by complex-type oligosaccharides (unit B) and high mannose-type oligosaccharides (unit A) prepared from porcine thyroglobulin. When thioglycolate-elicited macrophages were activated by lipopolysaccharide and/or the culture supernatant of concanavalin A-activated mouse spleen cells, they became tumoricidal and the number of the lectin-like molecules on the macrophage surface was found to increase. Since the binding and cytotoxic activities of these tumoricidal macrophages toward tumor cells were partially inhibited by D-galactose, the D-galactose-binding lectin-like molecules on the surface of tumoricidal macrophages might play an important role in macrophage-mediated cytotoxicity. These lectin-like molecules were then isolated from solubilized murine peritoneal exudate cells labeled with pyridoxal 5'-phosphate and sodium [3H]borohydride by affinity chromatography on columns of asialo unit B oligosaccharide-Sepharose 4B and/or beta-D-galactose-Bio-Gel P-100. The proteins bound to the asialo unit B oligosaccharide-Sepharose 4B column and eluted specifically were found to have approximate molecular weights of 79 000 and 18 000, and the protein bound to and eluted from the beta-D-galactose-Bio-Gel P-100 column had an approximate molecular weight of 77 000. These isolated proteins bound to the surface of glutaraldehyde-fixed tumor cells, and their binding was inhibited by D-galactose and also by D-mannose. Since most of the 77 kDa protein bound to the asialo unit B oligosaccharide-Sepharose 4B, this protein was assumed to be identical with the 79 kDa protein. These results suggest that the lectin-like molecules on murine macrophages have wide specificity and that one lectin-like molecule can bind both D-galactose and D-mannose.

Animals↗
PubMed1984

Structural changes of carbohydrate chains of human thyroglobulin accompanying malignant transformations of thyroid glands.

Carbohydrate moieties were released from human thyroglobulins prepared from normal and transformed, thyroid tissues by hydrazinolysis. The oligosaccharides thus prepared were fractionated by DEAE-cellulose, Bio-Gel P-4, concanavalin-A--Sepharose and Ricinus-communis-agglutinin--Sepharose columns and were characterized by exo-glycosidase and endo-glycosidase digestions, methylation analysis and 400-MHz 1H-NMR spectroscopy. These studies showed that the alterations accompanying malignancy occurred in complex-type carbohydrate chains and exhibited the following structural features: (a) a complex-type carbohydrate chain containing 1 phosphate in a diester linkage was present in all the malignant thyroglobulins. (b) Asialo-carbohydrate chains with biosynthetically incomplete structures were found in all of the malignant thyroglobulins. (c) Carbohydrate chains of higher molecular mass, which have repeating Gal-GlcNAc disaccharides and peripheral alpha-fucosyl residues were present in metastatic papillary carcinoma.

Carbohydrate Conformation↗
PubMed1984

Interaction of human platelet membrane glycoproteins with collagen and lectins.

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, an in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with 125I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.

Blood Platelets↗
PubMed1984

Macrophage-activating factor for cytotoxicity produced by a human T-cell hybridoma.

Human MAF-C (macrophage-activation factor for cytotoxicity)-producing hybridoma H2-E3-5 was prepared by somatic cell fusion of PHA-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphatic leukemia cells (CEM). The following activities were assayed: (1) macrophage-migration-inhibitory factor (MIF), (2) macrophage-activation factor for glucose consumption (MAF-G), (3) macrophage-activation factor for O2-formation (MAF-O), and (4) macrophage-activation factor for cytotoxicity (MAF-C). After anion-exchange chromatography, MAF-C activity could be distinguished from MIF and MAF-O activities. It is shown that MAF-C is not the same as MAF-G from the culture supernatants of CEM 11, a parent cell line of H2-E3-5. Furthermore, MAF-C from H2-E3-5 culture supernatants activated differentiated macrophages but not monocytes.

Antigens, Surface↗
PubMed1984

Characterization of mouse natural killer cell activating factor (NKAF) induced by OK-432: evidence for interferon- and interleukin 2-independent NK cell activation.

The bacterial immunopotentiator OK-432 induced natural killer cell activating factor (NKAF) from mouse spleen cells. OK-432-induced NKAF showed a single peak with an apparent mol. wt of 70 Kd by Sephadex G-100 chromatography and OK-432-induced interleukin 2 (IL-2) had the same mol. wt as NKAF. However, OK-432-induced interferon (IFN) showed molecular heterogeneity with two peaks at 90 Kd and 45 Kd. Further purification was achieved by Blue Sepharose affinity chromatography which copurified NKAF and IFN. The affinity-purified NKAF, however, was stable to heat (56 degrees C) and acid (pH 2) treatments. Moreover, anti-IFN failed to abolish NKAF activity and this activity was not absorbed by IL-2 dependent T cells. From isoelectric focusing analysis, a dissociation of NKAF and IFN was observed over the range of pI 6.5 to 8.0. Based on these results, KNAF appears to be a new kind of cytokine distinguishable from IFN and IL-2.

Animals↗
PubMed1984

Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature.

Nonsense mutations affecting the positive regulatory gene (htpR) of heat shock response have been obtained in a strain of Escherichia coli carrying no suppressor. The mutants can grow only at temperatures below 34 degrees C-35 degrees C. Heat, ethanol, and coumermycin induce major heat shock proteins in the wild-type but not in the htpR mutants. In contrast, the level of heat shock proteins synthesized at low temperature is unaffected. The htpR gene product is thus required for induction of heat shock proteins by heat or other stresses but not for their "basal-level" synthesis. Nucleotide sequence has been determined for the wild-type and the mutant alleles of htpR. The coding region appears to consist of 852 nucleotide pairs that correspond to 284 amino acids. Sequences commonly considered as signals for transcriptional initiation and termination were found flanking the coding region. Within this region, six amber, one opal, and two missense mutations were identified; the nonsense mutations are scattered along the gene, some being very close to the presumed amino terminus. These results indicate that the absence of htpR gene product is directly responsible for the failure to respond to heat shock or other stresses and for the inability to grow at high temperature. We propose that htpR represents a new class of genes that are essential for growth only at high temperatures (greater than 35 degrees C). Implications of the sequence homologies found among htpR, rpoD, and nusA proteins are discussed.

Aminocoumarins↗
PubMed1984

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma.

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.

Cell Line↗
PubMed1984

Establishment of a long-term cultured suppressor T-cell line specific for guanosine.

Induction of suppressor cells specific for guanosine in C57BL/6 mice with syngeneic spleen cells coupled with guanosine was demonstrated. Spleen cells from mice that had been tolerized by the injection of guanosine-coupled syngeneic spleen cells (G-SC) were repeatedly stimulated with mitomycin C-treated G-SC (MMC-G-SC) in vitro. These cells effectively suppressed the secondary in vitro response to guanosine-keyhole limpet haemocyanin (G-KLH) but did not suppress the response to sheep erythrocytes (SRBC). From these cell populations a long-term-cultured suppressor T-cell line specific for guanosine was established by using interleukin-2 and MMC-G-SC. This cell line suppressed the response to G-KLH but did not suppress the response to trinitrophenyl-KLH and SRBC. The suppressive effect was completely eliminated by treatment with anti-Thy 1.2 and complement.

Animals↗
PubMed1984

Separation and characterization of macrophage precursors and of interleukin 2-responding cells from nylon wool-nonadherent murine spleen cells by using Bauhinia purpurea agglutinin.

Precursors of macrophages were found to be enriched in nylon wool-nonadherent cells which are often used as purified T cells. By using a 2-step agglutination sedimentation method with Bauhinia purpurea agglutinin (BPA), the precursors of macrophages which were enabled to proliferate and differentiate by the addition of colony-stimulating factor (CSF) or high concentrations of concanavalin A (Con A) were exclusively separated into a nylon wool-nonadherent, BPA-agglutinated (bottom) fraction. Cells responsive to some kind of factor(s) other than CSF in Con A supernatants (Con A-Sup) were also fractionated into the bottom fraction. Under these conditions, T cells were separated into both BPA-nonagglutinated (top) and agglutinated (bottom) fractions. The proliferative response of the cells in the top fraction was higher than that in the bottom fraction when they were stimulated with an optimal concentration of Con A (3 micrograms/ml). As assessed by the Con A pulse experiments, T cells in the bottom fraction required larger amounts of interleukin 2 (I1-2) than those in the top fraction for proliferation and the growth of T cells in both fractions was completely dependent upon I1-2.

Adhesiveness↗
PubMed1984

Effect of a monoclonal anti-carcinoembryonic antigen antibody-ricin A-chain conjugate on human carcinoembryonic antigen-producing tumor cells.

For the development of therapeutic agents that are coupled to tumor specific carriers, an artificial protein hybrid conjugate (immunotoxin) containing monoclonal anti-carcinoembryonic antigen (CEA) antibody which binds specifically to the cell surface of CEA-producing tumor cells and a toxic subunit of ricin (A-chain) was prepared. This conjugate exerted strong cytotoxicity specifically toward CEA-producing tumor cells in vitro. In vivo, it has anti-tumor effects on ascitic tumor cells, and on free tumor cells before the solid tumor has been established. However, for the established solid tumor, a significant decrease of tumor growth was not seen even after repeated intravenous administrations of the antibody-ricin A-chain conjugate. Furthermore, the conjugate was found to be stable in both mouse and human serum in vitro, but after intravenous injection, it was inactivated in vivo quickly.

Animals↗
PubMed1983

Phospholipase A2 of chicken erythrocyte membranes.

Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins.

Animals↗
PubMed1983

Fractionation of mouse cytotoxic T cells by use of lectins.

Mouse cytotoxic T lymphocytes (CTLs), induced in vivo and in vitro in mixed-lymphocyte cultures, were fractionated into agglutinated and unagglutinated cells by use of various lectins. Cytolytic activity was enriched in the cell fraction agglutinated by various 2-acetamido-2-deoxy-alpha-D-galactopyranose-specific lectins, namely, Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), and Phaseolus limensis agglutinin (LBA). Only a little cytolytic activity remained in the unagglutinated cell fraction. Furthermore, the relationship between the binding of lectins and the cytolytic activity in various CTL cell lines was investigated with a fluorescence-activated cell sorter, and high-rank correlation was found between the cytolytic activity of these CTL cell-lines and the binding of DBA or HPA to them, but weaker rank correlation in the cases of LBA and peanut agglutinin (PNA). DBA was found to bind to almost all cells of interleukin-2-dependent CTL cell-lines tested. Although the DBA-positive CTL cell-lines have strong cytolytic activity, the DBA-negative cells, which consist of a small proportion of the CTLs, also exhibited cytolytic activity. Thus, the major part of CTLs has binding sites for alpha-D-GalNAc-specific lectins, particularly for DBA, and this lectin is useful for the enrichment of CTLs. However, the binding sites for DBA cannot be regarded as an exclusive differential marker for CTLs.

Animals↗
PubMed1983

Structural requirements for the binding of high-mannose-type glycopeptides to immobilized pokeweed Pa-2 lectin.

The structural requirements for the interaction of asparagine-linked glycopeptides with immobilized pokeweed mitogen Pa-2 were investigated. Some high-mannose-type glycopeptides obtained from porcine thyroglobulin were found to have strong affinity for Pa-2-Sepharose, whereas complex- and hybrid-type glycopeptides were shown to have much weaker affinity. The elution profiles of various glycopeptides modified by glycosidase treatment and acetolysis showed that the total structure alpha-D-Manp-(1 leads to 2)-alpha-D-Manp-(1 leads to 2)-alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-beta-GlcpNAc-(1 leads to 4)-beta-GlcpNAc leads to Asn was essential for the binding of glycopeptides to a Pa-2-Sepharose column.

Animals↗