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T Osawa

Publications and source records attributed to T Osawa.

At least 217 records · Page 12Linked to original sources
PubMed1991

Natural killer (NK) cell activating factor produced by a human T-cell hybridoma.

A human T-cell hybridoma (KC8-1.10), whose culture supernatant augments peripheral blood lymphocytes (PBL)-mediated spontaneous cytotoxicity against K562 cells, was established. This activity [natural killer (NK) cell activating activity] appears to be not due to interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) for the following reasons: 1) KC8-1.10 produced negligible or small amounts of IFNs and IL-2. 2) The NK cell activating activity in the KC8-1.10 culture supernatant was not neutralized by anti-IFN-gamma antiserum and stable even after pH 2 treatment for 24 hr, which is known to destroy IFN-gamma activity. 3) IL-2-dependent cell line absorbed IL-2 more efficiently than it absorbed the NK cell activating activity, and the latter activity was not retained by Blue Sepharose column in contrast with IL-2. The NK cell activating factor in the KC8-1.10 culture supernatant appears to be a glycoprotein, because the activity was abolished with pronase treatment or with boiling for 5 min and because the activity was retained by concanavalin A- and Pisum sativum agglutinin-agarose. Finally it was found that the NK cell activating activity requires Leu 11b+ cells to exert its effect.

Cytotoxicity, Immunologic↗
PubMed1991

Effect of cysteine on bovine serum albumin (BSA) denaturation induced by solar ultraviolet (UVA, UVB) irradiation.

Long-term exposure to natural sun-light (UVA, UVB) induced fluorescence and caused disulfide bond formation in bovine serum albumin (BSA). The addition of cysteine enhanced the bond formation to such an extent that a solution of BSA was transformed into an insoluble gel. The disulfide bonds in the gels are derived from internal-SH groups of protein. This reaction occurred even if cysteine was added after exposure to ultraviolet (UV)-irradiation. Fluorescent substances seem to be involved in this reaction. On the other hand, low concentrations of cysteine (less than 5 mM) inhibited both fluorescence and disulfide bond formation. The addition of glutathione to BSA produced the same effect as that of cysteine. The addition of thiourea to BSA solution inhibited fluorescence, but did not inhibit disulfide bond formation. We assume that external-SH compounds such as cysteine and glutathione, which have high reactivity with hydroxyl radicals (.OH), act not only as free-radical scavengers, but also as radical mediators in the polymerization of protein through disulfide cross-links induced by UV-irradiation. Solar UVA as well as UVB irradiation are shown to have the same effect on the protein polymerization.

Cysteine↗
PubMed1991

Mechanism of immunosuppression by a murine non-specific suppressor T cell line, SB-1.

The mechanism of immunosuppression by a murine non-specific suppressor T cell clone (SB-1), which was established from concanavalin A (Con A)-activated murine spleen cells [Jpn. J. Exp. Med., 53, 139 (1983)], was investigated. SB-1-induced decrease of antigen non-specific plaque-forming cell (PFC) response was restored by the addition of 5 x 10(-6) M indomethacin, an inhibitor of cyclooxygenase. On the other hand, suppressive activity for antigen non-specific PFC response was found in the culture supernatant of SB-1 cells in the presence of proteose peptone-elicited macrophages. This induction of the suppressive activity was also inhibited by 5 x 10(-6) M indomethacin. Furthermore, 10(-5) M prostaglandine E2 (PGE2) was found to induce suppressive activity in the culture supernatant of SB-1 cells. 10(-5) M PGE2 did not suppress interleukin-2 (IL-2)-dependent proliferation of SB-1 cells, but increased a small quantity of protein synthesis and deoxyribonucleic acid (DNA) synthesis of SB-1 cells in the absence of IL-2. These results suggest that a cyclooxygenase product(s) such as PGE2 from macrophages may induce production of soluble suppressor factors in SB-1 cells. To characterize these suppressor factors, those in the culture supernatant of PGE2-stimulated SB-1 cells were partially purified by ammonium sulfate fractionation and sequential column chromatographies on diethylaminoethyl (DEAE)-cellulofine and Sephacryl S-200. Suppressor activities were eluted at the positions corresponding to the molecular masses of 10, 30, 70 and over 70 kilodalton (kDa) on Sephacryl S-200 column chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

Amino Acid Sequence↗
PubMed1991

Macrophage migration inhibitory factor (MIF) produced by a human T cell hybridoma clone.

A macrophage migration inhibitory factor (MIF) was purified to homogeneity from the serum-free culture supernatant of a human T cell hybridoma clone called F5. This clone was established by means of somatic fusion, using the emetine-actinomycin D selection method, and produced a large amount of MIF. The MIF activity in the culture supernatant of F5 cells was not due to contaminating interferon-gamma (IFN-gamma), which is known to possess MIF activity. Furthermore, other known cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF), were also revealed to have MIF activity, but our MIF was different from these known factors. F5 cells produced two species of MIF that could be separated on a phenyl-Sepharose column. MIF-1 (the more hydrophilic species of the two) was purified to homogeneity by sequential hydrophobic chromatography, ion-exchange chromatography, dye ligand affinity chromatography, and high-performance liquid chromatography (HPLC) on ion-exchange and reverse-phase columns. Finally, 4600-fold enrichment, as to specific activity, of MIF-1 was achieved. The purified MIF-1 was digested with endoproteinase Lys-C into some peptide fragments and the amino acid sequences of the peptides obtained were determined. No sequence identity between our MIF-1 and other proteins was observed. Then, antibodies were raised against a peptide synthesized according to the determined amino acid sequence. They specifically reacted with MIF-1 and reduced its migration inhibitory activity. Based on these results, we conclude that the determined amino acid sequence was certainly that of MIF-1.

Amino Acid Sequence↗
PubMed1990

Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye.

To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KCl extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10(-7) and 10(-5) M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10(-6) and 10(-4) M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene.

Amino Acid Sequence↗
PubMed1990

Concanavalin A-induced receptor aggregation stimulates the tyrosine kinase activity of the insulin receptor in intact cells.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.

Animals↗
PubMed1990

Characterization through cDNA cloning of galactoprotein b3 (Gap b3), a cell surface membrane glycoprotein showing enhanced expression on oncogenic transformation. Identification of Gap b3 as a member of the integrin superfamily.

Galactoprotein b (Gap b) is a group of glycoproteins of fibroblast cell membranes showing enhanced expression in association with oncogenic transformation (Carter, W.G., and Hakomori, S. (1977) Biochem. Biophys. Res. Commun. 76, 299-308). We purified Gap b3 (one of the Gap b group) to homogeneity from hamster fibroblast NIL cells transformed with polyoma virus (NILpy). Partial amino acid sequence was determined, and the cDNA clones encoding this protein were isolated by oligodeoxynucleotide hybridization. The 5.0-kilobase pair cDNA encodes a protein of 1051 amino acid residues containing a signal peptide (32 residues), a large extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic domain (32 residues). The overall structure of Gap b3 is similar to that of the alpha subunit of so-called integrin receptors in the following respects: (i) presence of metal-binding sequences; (ii) location of a transmembrane domain near C terminus; (iii) matched positions of most of the cysteine residues. This, together with extensive amino acid sequence homology, strongly suggests that Gap b3 is a member of the integrin family. Gap b3 consists of two polypeptide chains (Mr = 110,000 and 30,000), which seem to be proteolytic cleavage products connected by disulfide bonds from a precursor protein. Southern hybridization shows the presence of a single copy gene in mice and humans as well. Northern hybridization analysis shows that the enhanced expression of Gap b3 in transformed cells is due to increased transcription of its message.

Amino Acid Sequence↗
PubMed1990

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages.

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.

Animals↗
PubMed1990

Nerve regeneration through the cryoinjured allogeneic nerve graft in the rabbit.

To examine whether the 3-4-cm-long allogeneic basal lamina tubes of Schwann cells serve as conduits for regenerating axons in rabbits, allogeneic saphenous nerve, which had been predenervated and pretreated by freezing, were transplanted from Japanese White rabbits (JW) to New Zealand White rabbits (NW). Animals were killed 1, 2, 6, 8, and 14 weeks after transplantation, and the cytology at the mid-portion of the grafts was examined by electron microscopy. The distal portion of the host saphenous nerves was also examined 14 weeks after grafting. Myelin sheath debris was phagocytosed by macrophages, while the basal lamina of Schwann cells were left intact in the form of tubes. Regenerating axons were first found in such basal lamina tubes 2 weeks after grafting, and gradually increased in number. Host Schwann cells accompanied the regenerating axons behind their growing tips, separating them into individual fibers and forming thin myelin sheaths on thick axons by 6 weeks after grafting. Regenerating nerves were divided into small compartments by new perineurial cells. Newly formed blood vessels were situated outside the compartment 8 weeks after grafting. The percentage of myelinated fibers in the regenerating nerves was roughly 10% at 8 weeks and 30% at 14 weeks after grafting. The diameter of the regenerating axons, both myelinated and unmyelinated, was less than that of normal axons at all the stages examined. Numerous regenerating axons, some of which were fully myelinated, were found at the site 10 mm distal to the distal end of the graft 14 weeks after grafting.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Structural requirements for the binding of oligosaccharides to immobilized lectin of Erythrina variegata (Linn) var. orientalis.

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins with Erythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100 degrees C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal sugar sequence, which is an I-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.

Amino Acid Sequence↗
PubMed1990

Carbohydrate binding specificities of several poly-N-acetyllactosamine-binding lectins.

The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with various N-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns. High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100 degrees C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal beta 1-4GlcNAc beta 1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to the Datura stramonium agglutinin (DSA)-, Lycopersicon esculentum (tomato) agglutinin- and Solanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc beta(1-6)- linkage for interaction.

Carbohydrate Metabolism↗
PubMed1990

Allogeneic nerve grafts in the rat, with special reference to the role of Schwann cell basal laminae in nerve regeneration.

The role of basal laminae as conduits for regenerating axons in an allogeneic graft was examined by transplanting a 3 cm long segment of the sciatic nerve from the Brown Norway to the Fischer 344 strain of rat. These strains are not histocompatible with each other. In order to compare the nerve regeneration in variously treated grafts, three different types of graft were employed: non-treated (NT), predenervated (PD), and predenervated plus freeze-treated (PDC) grafts. The cytology of nerve regeneration through these grafts was examined by electron microscopy at four, seven, 14, 30 and 60 days after grafting. In the PDC graft, in which Schwann cells were dead on grafting, basal laminae were well preserved in the form of tubes after Schwann cells and myelin sheaths had been removed at seven days after grafting. Regenerating axons accompanied by immature host Schwann cells grew out through such basal lamina tubes in the same fashion as observed in our previous studies. By day 14, axons extended as far as the middle of the graft. In the proximal part they were separated into individual fibres and even thinly myelinated by Schwann cells. On the other hand, in the NT and PD grafts in which Schwann cells were alive on grafting, most Schwann cells and myelin sheaths appeared to undergo autolytic degeneration by day 14, while Schwann cell basal laminae were left almost intact in the form of tubes. A few regenerating axons were seen associated with Schwann cells in the proximal portion by day seven. It is probable that host Schwann cells moved into the graft after donor cells had been degraded. Schwann cell basal laminae tended to be damaged at the site of extensive lymphoid cell infiltration. By day 30, regenerating axons had arrived at the distal end of the graft in all three types of graft: in the PDC graft thick axons were fully myelinated, whereas in the PD graft they were only occasionally myelinated and in the NT graft most axons were still surrounded by common Schwann cells. By 60 days after grafting, regenerating axons were well myelinated in the host nerve as observed 1 cm distal to the apposition site in all the three types of graft. These findings show that Schwann cell basal laminae can serve as pathways (most efficiently in the PDC graft) for regenerating axons in a 3 cm long allograft in the rat.

Animals↗
PubMed1990

Elevation of the activities of glycosyl transferases involved in polylactosaminoglycan biosynthesis in autoimmune MRL lpr/lpr mouse T cells.

Lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-(+)/+ (+/+) mice were compared as to glycosyl transferase activities involved in the biosynthesis of polylactosaminoglycans. The N-acetylglucosaminyl transferase (GlcNAc transferase) activity responsible for the extension of polylactosaminoglycans was assayed. The reaction products with this GlcNAc transferase were characterized by sequential glycosidase treatment and methylation analysis, and the enzyme was found to be classifiable as an UDP-GlcNAc:N-acetyllactosamine beta 1-3 GlcNAc transferase (polylactosamine extension enzyme). The activity of this GlcNAc transferase in T cells from enlarged LN of lpr mice was 3-6 times higher than that in T cells from +/+ mice. On the other hand, activated T cells from +/+ mice only showed about a 2-fold increase in the activity of the transferase, compared with that in resting T cells. B cells from +/+ mice also showed a significantly higher activity of the transferase than +/+ T cells, the enzyme activity being comparable to or slightly lower than that in lpr T cells. Furthermore, when the reaction mixture contained both UDP-GlcNAc and UDP-Gal as donors, extension of the Gal-GlcNAc residue was observed. These results indicated the biosynthetic basis for the abundance of polylactosaminoglycans in lpr T cells and normal B cells. We also found that lpr T cells exhibited significant UDP-GlcNAc:asialo-bovine submaxillary mucin GlcNAc transferase activity. Only weak activity of this enzyme was detected in +/+ resting and activated T cells, and B cells. This enzyme activity suggested the potential for polylactosaminoglycan formation on the mucin-type sugar chains on the surface of lpr T cells.

Amino Sugars↗