[Large scale production of immune cell producing factors and biotechnology].
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Biomedical Engineering↗
Biomedical subjects
T Osawa
Publications and source records attributed to T Osawa.
Sensitivity of pre-T cells to natural thymocytotoxic autoantibodies developed in New Zealand Black mice.
The effect of affinity purified natural thymocytotoxic autoantibodies from New Zealand Black (NZB) mice (NTA-1 and NTA-2) against pre-T cells was studied. Pre-treatment of spleen cells from nu/nu mice or bone marrow cells from DBA/2 mice with NTA-1 and complement (C) or with NTA-2 and C markedly inhibited the induction of T cell markers with a thymic extract in vitro. The effect of NTA-2 and C was abolished by absorption of NTA-2 with neuraminidase treated thymocytes, whereas its effect was not abolished by absorption with intact thymocytes. The effect of NTA-1 and C was abolished by absorption of NTA-1 with either neuraminidase treated thymocytes or intact thymocytes. The effect of NTA-2 was inhibited by lactose whereas the effect of NTA-1 was not. These results suggest that NTA-2 specifically recognizes the cell surface characteristics of pre-T cells and blocks the differentiation pathway of T cells.
Animals↗
Human T cell hybridomas producing lymphokines. I. Establishment and characterization of human T cell hybridomas producing lymphotoxin and migration inhibitory factor.
Stable human T cell hybridomas were produced by cell fusion between PHA-activated human peripheral blood lymphocytes and human acute lymphatic leukemia cells of a CEM cell line whose proliferation had been inhibited by treatment with an irreversible protein synthesis inhibitor (emetine) and an irreversible RNA synthesis inhibitor (actinomycin D). Two hybrid cell lines (E10, F8) thus produced expressed OKT3-reactive antigen, HLA-A1 and -B8 antigens derived from CEM cells, and HLA-A2 antigen derived from PBL, and they have 10 more chromosomes than CEM cells. Furthermore, these cell lines continuously secreted lymphotoxin over 3 mo. E10 was found to produce MIF in addition to lymphotoxin. Cloning of E10 and the relationship between the functions and surface phenotypes of E10 sublines were examined. Lymphotoxin-producing hybrids (E10-15 and E10-37) express more OKT8-reactive antigen than OKT4-reactive antigen, while lymphotoxin and MIF-producing hybrids (E10-25, E10-42, and E10-43) express more OKT4-reactive antigen than OKT8-reactive antigen.
Antibodies, Monoclonal↗
[Cell membrane proteins (author's transl)].
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Actins↗
Inhibitory activity of disaccharide-L-asparagine compounds against hemagglutination by various lectins.
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Acetylglucosamine↗
The carbohydrate moiety of band 3 glycoprotein of human erythrocyte membranes. Structures of lower molecular weight oligosaccharides.
Band 3 glycoprotein purified from human blood group A erythrocyte membranes was subjected to hydrazinolysis to release carbohydrate moieties. The neutral oligosaccharide fraction, which constituted more than 85% of the total carbohydrates released, was fractionated by gel chromatography on a column of Sephadex G-50 into two fractions, i.e. higher molecular weight and lower molecular weight fractions. From the lower molecular weight fraction, three oligosaccharides were obtained by chromatographies on columns of Bio-Gel P-4, concanavalin A-Sepharose, and lentil lectin-Sepharose. Their structures were investigated by a combination of compositional analyses, glycosidase treatments, methylation and Smith periodate degradation, and proposed to be: Gal beta 1 - 4GlcNAc beta 1 - 2Man alpha 1 - 6(GlcNAc beta 1 -4) (Gal beta 1 - 4GlcNAc beta 1 - 2Man alpha 1 - 3)Man beta 1 - 4GlcNAc beta 1 - 4(Fuc alpha 1 - 6)GlcNAc, Gal beta 1 - 4GlcNAc beta 1 - 2Man alpha 1 - 6(Gal beta 1 - 4GlcNAc beta 1 - 2Man alpha 1 - 3)Man beta 1 - 4GlcNAc beta 1 - 4(Fuc alpha 1 - 6)GlcNAc, and GlcNAc beta 1 - 2Man alpha 1 - 6(GlcNAc beta 1 - 4) (Glc NAc beta 1 - 2Man alpha 1-3) Man beta 1 - 4GlcNAc beta 1 - 4(+/-Fuc alpha 1 - 6)GlcNAc. It was also demonstrated in this study that most of the higher molecular weight oligosaccharides, whose peripheral structure had previously been reported (Tsuji, T., Irimura, T., and Osawa, T. (1980) Biochem. J. 187, 677-686), also contained the same core hexasaccharide structure, Man alpha 1 - 6(Man alpha 1-3)Man beta 1 - 4GlcNAc beta 1 - 4(Fuc alpha 1-6)GlcNAc, as that of the lower molecular weight oligosaccharides.
Anion Exchange Protein 1, Erythrocyte↗
Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized wheat germ agglutinin.
The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins with wheat germ agglutinin (WGA) were investigated by using affinity chromatography on a WGA-Sepharose column. So-called hybrid-type glycopeptides obtained from ovalbumin [Yamashita, K., Tachibana, Y., & Kobata, A. (1978) J. Biol. Chem. 253, 3862--3869] were found to have high affinity for WGA--Sepharose, whereas high mannose-type and complex-type glycopeptides were shown to have low affinity. The elution profiles of various glycopeptides modified by glycosidase treatment, Smith periodate degradation, acetolysis, and hydrazinolysis showed that the GlcNAcbeta 1--4Manbeta 1--4GlcNAc beta 1--4GlcNAc-Asn structure was essential for the binding of glycopeptides to a WGA-Sepharose column. Thus, it was revealed that both the N,N'-diacetylchitobiose moiety and the beta-N-acetylglucosaminyl residue linked to C-4 of the beta-linked mannose residue contributed to the interaction of the glycopeptide with WGA-Sepharose. The substitution at C-6 of the innermost beta-N-acetylglucosaminyl residue by an alpha-fucosyl residue or at C-6 of the beta-linked mannose residue by another mannose residue in the above structure reduced the affinity of glycopeptides for the column.
Chromatography, Affinity↗
Structure of carbohydrate unit A or porcine thyroglobulin.
The unit A-type glycopeptides were purified from porcine thyroglobulin by Pronase digestion followed by chromatography on a DEAE-Sephadex A-25 column. These glycopeptides were separated into five fractions (UA-I, -II, -IV and -V) by Dowex 50W (X2) column chromatography. Fractions UA-I, -II, -III, -IV and -V were found to have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, (Man)7(GlcNAc)2-Asn, (Man)6(GlcNAc)2-Asn and (Man)5(GlcNAc)2-Asn respectively. The structures of these five fractions were investigated by the combination of exo- and endo-glycosidase digestions, methylation analysis. Smith periodate degradation and acetolysis. The results showed that fraction UA-V had the simplest structure: see formula in text. The larger glycopeptides (fractions UA-I, -II, -III and -IV) contained additional mannose residues alpha (1 leads to 2)-linked to the terminal mannose residues in the above core structure. These unit A-type glycopeptides appear to be biosynthetic intermediates that are to be processed to form complex-type glycopeptides (unit B-type sugar chains).
Animals↗
The structure of carbohydrate unit B of porcine thyroglobulin.
The oligosaccharide fraction was obtained from porcine thyroglobulin by hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by successive chromatographies on columns of DEAE-cellulose and concanavalin A-Sepharose, and their structures were investigated by the combination of endo- and exo-glycosidase digestions, methylation analysis and Smith degradation. From the results of these studies, the structures of the unit B oligosaccharides were proposed to be as follows: see formula in text. Thus the glycoprotein was found to have triantennary and biantennary complex-type oligosaccharides as acidic sugar chains. Concerning the triantennary oligosaccharides, the following structural features were shown: (1) the sialic acid residues were not localized on certain specific branches but distributed on all three branches; (2) however, alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the terminal of the branch arising from C-4 of the branching alpha-mannose residue, whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of the other branches; (3) the outer branching alpha-mannose residue was attached to C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were observed to exist.
Animals↗
Structure of a complex-type sugar chain of human glycophorin A.
Tryptic glycopeptide T1 of human glycophorin A [Tomita, M., & Marchesi, V. T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968] was subjected to hydrazinolysis. After N-acetylation, the complex-type oligosaccharides were isolated by gel filtration. The major neutral oligosaccharide (2000 molecular weight) was purified by a combination of ion-exchange and gel-permeation chromatography. Treatments with endo- and exo-glycosidases, periodate oxidation, and methylation analysis indicated that the major neutral oligosaccharide has the following structure: Gal beta 1 leads to -4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(GlcNAc beta 1 leads to 4)(Gal beta 1 leads to -4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4-(Fuc alpha 1 leads to 6)GlcNAc. This oligosaccharide was retained by Ricinus communis agglutinin-Sepharose and retarded by Sepharose 4B coupled with erythroagglutinating E4 isolectin from Phaseolus vulgaris. Retention by concanavalin A-Sepharose was observed only after treatment of the oligosaccharide with beta-galactosidase.
Carbohydrate Conformation↗
Effects of reduced amount of RNA polymerase sigma factor on gene expression and growth of Escherichia coli: studies of the rpoD450 (amber) mutation.
A mutant of Escherichia coli K-12 carrying an amber mutation (rpoD40) in the structural gene for RNA polymerase sigma factor and a temperature-sensitive amber suppressor (supF-Ts6) grows virtually normally at 30 degrees C, but does not grow at 42 degrees C due to the inability to synthesize sigma polypeptides (Osawa, T. and Yura, T., Mol Gen Genet 180, 293 - 300, 1980). When the mutant cells are transferred from 30 to 42 degrees C, the cellular amount of sigma relative to total protein is found to decrease from 50% (at 30 degrees C) to 10% of the wild-type level after about 2 h. The decrease of sigma is accompanied by a gradual decrease in RNA and protein syntheses and a sudden loss of viability. At the highest temperature (36 degrees C) that permits steady growth of this mutant, the amount of sigma and the growth rate become 6% and 50 to 60% of the wild type, respectively. These results suggest that the minimum level of sigma required for growth is 0.02 to 0.04 in terms of molar ratio of sigma to core enzyme, that is 6 to 10% of the wild type. Two-dimensional gel electrophoresis of proteins synthesized under the reduced sigma level reveals either markedly increased or decreased syntheses of several polypeptides, while no detectable effect is observed in the majority of polypeptides. Notably, the synthesis of a set of major heat-shock polypeptides is greatly enhances. Hence, the decrease of RNA polymerase holoenzyme relative to the core enzyme seems to affect the synthesis of individual proteins differentially, primarily at the level of transcription. The expression of the groE operon, one of the major heat-inducible operons in E. coli is also studied in some detail.
Escherichia coli↗
Purification and characterization of a catalytic subunit of an adenosine 3':5'-monophosphate-dependent protein kinase from human erythrocyte membranes.
Protein kinase [EC 2.7.1.37] of human erythrocyte membranes was solubilized with 0.5 M NaCl in 5 mM phosphate buffer, pH 6.7 at 4 degrees C and purified on a CM-Sephadex C-50 column, followed by affinity chromatography on a histone-Sepharose 4B column. The purified protein kinase gave a single band (molecular weight; 41,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 8.0 and a millimolar range of concentration of Mg2+ was required for its maximum activity. Histone and protamine were well phosphorylated by the protein kinase but casein and phosvitin were poor phosphate acceptors for the enzyme. The enzymic activity was not stimulated by cyclic AMP (cAMP). A cAMP-finding protein from human erythrocyte membranes inhibited the activity of the protein kinase, but the activity was restored with cAMP. A heat stable protein inhibitor from rabbit skeletal muscle also inhibited this enzyme. From these observations, this protein kinase seemed to be a catalytic subunit of the membrane bound cAMP-dependent protein kinase. This enzyme was strongly inhibited with Ca2+ in the presence of 1 mM MgCl2. Various sulfhydryl reagents and polyamines also had inhibitory activity on the protein kinase. Natural substrates of the enzyme were investigated using heat treated membranes and 0.5 M NaCl extracted membrane residues. Band 4.1, 4.2, and 4.5 proteins were phosphorylated but band 2 (spectrin) and band 3 proteins were poor substrates for this protein kinase.
Carrier Proteins↗
Mechanism of the concanavalin A-induced change of membrane fluidity of chicken erythrocytes.
When chicken erythrocytes labeled with a stearic acid derivative spin label was treated with concanavalin A (Con A), ESR spectra showed a change in the peaks due to the labels in membrane lipids, indicating an increase of membrane lipid fluidity. Addition of Con A increased the fluidity rapidly. This change was reversible only up to 30 min after adding Con A, and thereafter it gradually became irreversible. However, if the erythrocytes were treated with cytochalasin B and methyl alpha-D-mannoside, a complete return of fluidity to the normal level could be observed at any stage after the binding of Con A. The observation of freeze-fracture replicas of erythrocyte membranes by transmission electron microscopy also showed that the redistribution of intramembranous particles gradually became irreversible after exposure to Con A. These results suggest that the microfilament-like system, which modulates the distribution of cell surface receptors for Con A, participates in the modulation of membrane fluidity. Phospholipid methylation of chicken erythrocyte membrane was stimulated immediately after the binding of Con A. A methyltransferase inhibitor, 5'-deoxy-5'-S-isobutyl adenosine, abolished the increase of membrane fluidity within the first 10 min and also that occurring later than 60 min after adding Con A, but it was without effect on the elevated fluidity found between 20 and 60 min. Removal of extracellular Ca2+ had an inhibitory effect on the lasting increase of fluidity. These results suggest that the first increase of membrane fluidity by Con A may be caused by phospholipid methylation, while the second increase may depend on the rearrangement of Con A receptor glycoproteins through cross-linking wit Con A. The irreversible part of the membrane fluidity increase probably depends on Ca2+ influx, phospholipid methylation, and peripheral membrane proteins which constitute the microfilament-like system at the membrane inner surface.
Animals↗
Purification and characterization of a Cytisus-type Ulex europeus hemagglutinin II by affinity chromatography.
Ulex europeus hemagglutinin II [Cytisus-type anti-H(O) hemagglutinin] inhibited most by di-N-acetylchitobiose has been purified by affinity chromatography on a column of chitobiose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. The purified hemagglutinin was homogeneous by ultracentrifugal analysis and gave a single band by electrophoresis on polyacrylamide gel, and had a molecular weight of 105 000 by sedimentation equilibrium and an isoelectric point of pH 6.66. This hemagglutinin was found to be composed of four, apparently identical, subunits of a molecular weight of 25 000 +/- 2 000 by dodecyl sulphate-polyacrylamide gel electrophoresis, and to contain 10.3% carbohydrate in which mannose (3.7%) was the predominant sugar, with smaller amounts of glucose, glucosamine, xylose, fucose and galactose. Amino acid analysis of the purified hemagglutinin II showed a large amount of aspartic acid and serine, but as little as 0.1 mol/100 mol of cystine or methionine could be detected.
Amino Acids↗
Isolation and characterization of a mitogenic substance for murine and human B lymphocytes from Ulex europeus seeds.
A mitogenic substance, which stimulates murine and human B lymphocytes, has been isolated from the seeds of Ulex europeus. The m.w. of this mitogen was estimated to be approximately 100,000 by sodium dodecyl sulfate disc gel electrophoresis. Under physiologic conditions Ulex mitogen was in the form of a polymer. The isolated mitogen was found to contain 80% carbohydrate, in which glucose, galactose, and rhamnose were the predominant sugars. The carbohydrate portion of the mitogen could be responsible for the mitogenic activity, since the activity was destroyed by periodate treatment but not by protease digestion. It is unlikely that the mitogenic activity is due to the contamination by lipopolysaccharide (LPS), because Ulex mitogen strongly activated spleen cells from C3H/HeJ mice and did not contain a detectable amount of fatty acid. Spleen cells from nude mice, T-depleted spleen cells from conventional mice, or T-depleted spleen cells from conventional mice, or T-depleted lymphocytes from human peripheral blood responded as strongly as lymphocytes (T + B) from normal origin as measured by 3H-thymidine incorporation. It seems that the mitogenic effect is primarily on B cells. Ulex mitogen also induced the increase of immunoglobulin synthesis in murine and human B cells.
Animals↗
The structure of the dentine of sperm whales.
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Animals↗
The structure and apparent location of calcified nodules in the dentine of sperm whales.
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Animals↗
Immunopotentiation of tumor-bearing and toxohormone treated mice by human ceruloplasmin.
Human ceruloplasmin was shown to have a restorative effect on the decrease of delayed hypersensitivity in sarcoma-180-bearing mice and of helper T-cell activity in cell-free. Ehrlich cancerous ascitic fluid-treated mice. Furthermore, it was also shown that ceruloplasmin augments the in vivo generation of alloreactive cytotoxic cell which consist of cytotoxic T-lymphocytes, cytotoxic macrophages and K cells.
Adjuvants, Immunologic↗