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T Osawa

Publications and source records attributed to T Osawa.

At least 253 records · Page 14Linked to original sources
PubMed1989

Sugar binding specificities of anti-H(O) lectins disclosed by use of fucose-containing human milk oligosaccharides as binding inhibitors.

The binding to normal and sialidase-treated human erythrocytes of six 125I-labeled lectins [Ulex europeus lectin I (UEA-1) and II (UEA-II), Laburnum alpinum lectins I (LAA-I) and II (LAA-II), and Cytisus multiflorus lectins I (CMA-I) and II (CMA-II)], was studied in detail. Quantitative inhibition assays of the lectin binding to the cells were also performed with various human milk oligosaccharides as inhibitors. Based on a comparison of the inhibition constants of the inhibitors thus obtained with the association constants of the lectins to the cells, the relative activities of cell surface blood group antigens toward the lectins are discussed.

Carbohydrate Metabolism↗
PubMed1989

Partial purification of a macrophage-activating factor for glucose consumption (MAF-G) produced by a human T-cell hybridoma and its relation to a growth-promoting factor.

A material that enhances the glucose consumption of oil-induced peritoneal macrophages of guinea pig, the activity being named MAF-G activity, was purified approximately 780-fold from human T cell hybridoma conditioned medium. A material showing growth-promoting activity was co-purified with that showing the MAF-G activity from the same conditioned medium. The two materials showed an identical isoelectric point of pH 4.7. When the partially purified MAF-G increased glucose consumption, it did not induce glucose carbon-1 oxidation. MAF-G activity was inhibited by mitomycin C and colchicine, which inhibit DNA synthesis and mitosis, respectively, but not by 2-deoxy-D-glucose, an inhibitor of glucose metabolism. The partially purified MAF-G also enhanced the glucose consumption and proliferation of human monocytes. These observations suggest that MAF-G may be identical with the growth-promoting factor for macrophages.

Animals↗
PubMed1988

Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli.

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.

Amino Acid Sequence↗
PubMed1988

Carbohydrate-binding specificities of five lectins that bind to O-Glycosyl-linked carbohydrate chains. Quantitative analysis by frontal-affinity chromatography.

The carbohydrate-binding specificities of lectins purified from Agaricus bisporus (ABA-I), Arachis hypogaea (PNA), Bauhinia purpurea (BPA), Glycine max (SBA), and Vicia villosa (VVA-B4) have been studied by affinity chromatography on columns of the immobilized lectins, and quantitatively analyzed by frontal affinity chromatography. These five lectins could be classified into two groups with respect to their reactivities with typical mucin-type glycopeptides, beta-D-Galp-(1----3)-alpha-D-GalpNAc-(1----3)-Ser/Thr (2) and alpha-D-GalpNAc-(1----3)-Ser/Thr (3). One group, which consists of ABA-I, PNA, and BPA, preferentially binds to 2, and the other, which consists of SBA and VVA-B4, shows higher affinity for 3 than for 2. Among the lectins tested, only ABA-I was found to bind to a sialylated glycopeptide, whic which was prepared from human erythrocyte glycophorin A and contains three three tetrasaccharide chains having the structure of alpha-NeuAc-(2----3)-beta-D-GAlp-(1----3)-NeuAC-(2----6)]-alpha-D-Galp NAc-(1----, with an association constant of 15 microM, whereas the association constants of the other four lectins for this sialylated glycopeptide were less than 3.5 mM. On the other hand, removal of the beta-D-galactopyranosyl group from a glycopeptide containing sequence 2 resulted in decreased association constants for the three lectins of the first group, especially ABA-I and PNA. The two lectins of the second group showed a high affinity for 3, but SBA preferentially interacted with oligosaccharides containing the alpha-D-GalpNAc-(1----3)-beta-D-Galp-(1----3)-D-GlapNAc sequence, prepared from a blood group A-active oligosaccharide.

Carbohydrate Sequence↗
PubMed1988

Possible mechanism of inhibition of experimental pulmonary metastasis of mouse colon adenocarcinoma 26 sublines by a sialic acid: nucleoside conjugate.

As described previously (I. Kijima-Suda et al., Cancer Res., 46: 858-862, 1986), a sialyltransferase inhibitor, 5-fluoro-2',3'-isopropylidene-5'-O-(4-N-acetyl-2,4-dideoxy-3,6,7,8-tetra -O- acetyl-1-methoxycarbonyl-D-glycero-alpha-D-galactooctapyranosyl)ur idine (KI-8110), inhibits pulmonary metastasis of murine colon adenocarcinoma 26 sublines of high (NL-17) and low (NL-44) metastatic potential. To investigate the mechanism of this inhibition, the effect of KI-8110 on the metastatic cascade, especially on the interaction between tumor cells and platelets which may play a crucial role in tumor cell metastasis, was examined. NL-17 cells induced irreversible platelet aggregation in heparinized human platelet-rich plasma in vitro. This activity was reduced by pretreatment of the tumor cells with KI-8110. Inhibition of aggregation was also induced by the treatment of tumor cells with neuraminidase or Limax flavus agglutinin, a lectin specific for sialic acid. Sialic acid, fucose, sialyllactose, and bovine submaxillary mucin inhibited this tumor cell-induced platelet aggregation, while galactose, mannose, lactose, alpha 1-acid glycoprotein, fetuin, and asialo-bovine submaxillary mucin did not. KI-8110 also inhibited platelet-derived growth factor-dependent growth of NL-17 cells, but showed no effect on insulin or epidermal growth factor-dependent growth of the tumor cells. Platelet-derived growth factor-induced phosphorylation of membrane protein was reduced by treatment of NL-17 cells with KI-8110. The same result was obtained in the neuraminidase-treated membrane fraction of NL-17 cells. These results suggest that KI-8110 inhibits experimental tumor cell metastasis by inhibiting the interaction between tumor cells and host platelets in at least two pathways, and this may be due to a reduction of sialic acid contents of the membrane surface of tumor cells.

Animals↗
PubMed1988

The separation of immunocyte subpopulations by use of various lectins.

The use of lectins for the enrichment of various lymphocyte subpopulations was investigated. Bauhinia purpurea lectin (BPA) was found to be effective for the enrichment of B cells, and the B cells thus obtained were further fractionated with lentil lectin into subsets showing high and low responses to dextran sulfate. The ability of various lectins to selectively induce suppressor T cell activity or helper T cell activity was also examined. The suppressor T cells thus induced were enriched peanut lectin and, conversely, the helper T cells were enriched with Limulus polyhemus lectin. This method was applied to analysis of age-dependent changes in the levels of suppressor and helper T cells in autoimmune-prone mice. Cytotoxic T cells induced in an allogeneic mixed lymphocyte culture were enriched with Dolichos biflorus lectin (DBA). These cytotoxic T cells showed a specific killing effect in vitro. However, when spleen cells of tumor-bearing mice were fractionated by use of DBA, the DBA- cells mediated the regression of the tumors in vivo. BPA was also found to be effective for the enrichment of the interleukin-2-producing T cell subset and macrophage precursor cells. Using this technique, bone marrow cells of autoimmune-prone MRL/1 mice were found to be rich in macrophage precursor cells.

Animals↗
PubMed1988

A human macrophage hybridoma producing a cytotoxic factor distinct from TNF, LT, and IL-1.

A stable human macrophage hybridoma was established by somatic cell fusion between human peripheral blood monocyte-derived macrophages and an 8-azaguanine resistant clone of a human histiocytic lymphoma cell line U-937 (clone U-937-F9). The hybrid cell line (F9P) exhibited typical macrophage-like morphology and had 30 more chromosomes than U-937-F9 cells. Its macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mo-2 and LEU-M3 antigens on the cell surface, and the demonstration of phagocytic activity. Furthermore, when stimulated with lipopolysaccharide (LPS), this cell line could secrete a considerable amount of a cytotoxic factor (CTF). Distinct from the hybrid cell line, the parental U-937-F9 cells expressed neither Mo-2 nor LEU-M3 antigens on the cell surface, did not show phagocytic activity, and their culture supernatants did not show cytotoxic activity even after LPS stimulation. The activity of CTF in the culture supernatant of the LPS-stimulated hybrid cells could not be neutralized with anti-tumor necrosis factor, anti-interleukin-1, or anti-lymphotoxin antibodies. The CTF had a relative molecular mass of 45-60 x 10(3) daltons as determined by gel filtration on a column of Superose 12, and an isoelectric point of 5.1. The cytotoxic activity was also induced when the hybrid cells were stimulated with the concentrated supernatants of a human T-cell hybridoma containing macrophage activating factor for cytotoxicity or with LP3 tumor cells which were used as target cells.

Antibodies↗
PubMed1988

A re-evaluation of the cytology of cat Pacinian corpuscles. I. The inner core and clefts.

The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristics concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.

Animals↗
PubMed1988

Enhancement of the activities of glycosyltransferases involved in the biosynthesis of mucin-type sugar chains 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 their cell surface lectin-binding sites and glycosyltransferase activities. T cells from enlarged LN of lpr mice expressed a higher amount of binding sites for lectins reactive to mucin-type sugar chains than normal +/+ mouse T cells. Correspondingly, glycosyltransferase activities involved in the biosynthesis of mucin-type sugar chains were higher in lpr mouse T cells than in +/+ T cells. The activities of UDP-N-acetylgalactosamine (GalNAc):polypeptide GalNAc transferase and UDP-galactose (Gal):asialo bovine submaxillary mucin (BSM) Gal transferase were found to be elevated. The activity of UDP-Gal:asialo-agalacto transferrin Gal transferase, which is involved in the biosynthesis of complex type sugar chains, was also increased in lpr mice but to a smaller extent than the mucin-type Gal transferase activities. An abnormality in sialyltransferase activity was also found in lpr T cells.

Animals↗
PubMed1988

Functional deficiencies of spleen dendritic cells in autoimmune MRL/lpr mice.

We investigated functional aspects of splenic dendritic cells (DC) obtained from MRL/MpJ-lpr/lpr (MRL/l) mice, and their normal counterparts, MRL/MpJ-+/+ (MRL/+) mice. In vitro antigen-presenting activity of DC obtained from MRL/l mice was impaired compared with that of DC from MRL/+ mice. Another major functional aspect of DC, i.e., stimulator cell activity in the allogeneic mixed lymphocyte reaction (MLR), was also found to be impaired in MRL/l mice. MRL/l mice and MRL/+ mice were also examined for co-operation between macrophages (M luminal diameter) and DC. We found peritoneal resident M luminal diameter obtained not only from MRL/+ mice but also from MRL/l mice released a soluble factor which enhanced the activity of DC from the respective mice. Furthermore, DC from both MRL/l and MRL/+ mice responded to the culture supernatant of a mouse macrophage hybridoma clone, S44, which releases a factor that enhances the antigen-presenting activity of DC, and their activity as stimulator cells in the allogeneic MLR was enhanced. However, the degree of enhancement was less in MRL/l mice than in MRL/+ mice.

Animals↗
PubMed1988

Effect of an autoreactive T cell clone from (NZB x NZW)F1 mice on the production of anti-DNA antibodies in vivo and in vitro.

An autoreactive T lymphocyte clone, designated as F1C4 was established from an autoimmune mouse strain (NZB/NZW)F1. This clone proliferated in the presence of mitomycin C-treated splenic adherent cells (MMC-SAC) from syngeneic mice. This response was dependent on the numbers of MMC-SAC. The specificity of F1C4 for I-A was determined by an inhibition test carried out with monoclonal anti-Ia sera. Furthermore, the F1C4 cells did not exhibit alloreactivity in a proliferation assay and did not react to foreign antigens such as fetal calf serum (FCA) used in the culture medium. When F1C4 cells were cultured with autologous non-T cells in the absence of antigen, they strongly enhanced IgM class anti-ssDNA production from non-T cells of both young and old B/W F1 mice at appropriate cell numbers in vitro. Furthermore, the production of IgG class anti-ssDNA from non-T cells of old B/W F1 mice was also enhanced. The adoptive transfer of F1C4 cells enhanced the levels of both IgM and IgG anti-ssDNA antibodies in the serum of aged B/W F1 mice. Moreover, the serum levels of anti-ssDNA of IgG2a and IgG2b subclasses were readily enhanced by the transfer of F1C4 in vivo.

Animals↗
PubMed1988

Hygroscopicity of cefazolin sodium: application to evaluate the crystallinity of freeze-dried products.

The hygroscopicity behavior of the pentahydrate, monohydrate, amorphous, and dehydrated forms of solid cefazolin sodium (CEZ) was studied under different relative humidity (RH) conditions. Between 42 and 86% RH, the pentahydrate (alpha form), the monohydrate, and their dehydrated forms absorbed atmospheric moisture equivalent to their hydrate numbers. The pentahydrate demonstrated a hysteresis effect at 15 and 31% RH. On the other hand, the water content of the amorphous form increased linearly with increases in RH. The noncrystalline state was maintained below 56% RH. For the dehydrated alpha form there was a distinct birefringence when viewed under polarizing light, the X-ray diffraction pattern was weak and diffuse, and the infrared (IR) spectra were discernibly different from that of the amorphous form. The freeze-dried CEZ showed hygroscopic behavior almost similar to that of the dehydrated alpha form. Two-component mixtures of various CEZ forms showed a linear relationship between the water content and the mixing ratio when stored at 31, 42, and 56% RH. From the hygroscopicity data, the crystallinity of freeze-dried CEZ could be estimated as the percentage of the dehydrated alpha form.

Adsorption↗
PubMed1988

Estimation of the degree of crystallinity of cefazolin sodium by X-ray and infrared methods.

The pentahydrate (alpha form) of cefazolin sodium (CEZ) exhibited sharp X-ray diffraction peaks, while the dehydrated alpha form showed weak but distinct diffraction peaks. As expected the amorphous form exhibited a diffuse and halo diffraction pattern. The X-ray procedure to estimate the degree of crystallinity of CEZ was based upon the measurement of the total scattering and the scattering from the crystalline region of the drug. The major difference in the infrared (IR) spectra among the three forms of CEZ was the absence of a spectral band at 1542 cm-1 in the amorphous form. The IR procedure was based upon the measurement of the peak percentage area ratio between the bands at 1542 and 1760 cm-1, where the latter was used as a normalizing peak. The degree of crystallinity of CEZ samples, obtained by either freeze-drying aqueous CEZ solutions or storing the crystalline forms under different humidity conditions, was determined by these two methods. Although the correlation of results by the two methods was good, the X-ray procedure appears to be superior since it can differentiate among the three solid CEZ forms, whereas IR could distinguish between only crystalline and amorphous CEZ, reproducibly.

Cefazolin↗
PubMed1988

Properties of a novel GTP-binding protein which is associated with soluble phosphoinositides-specific phospholipase C.

Recently we demonstrated the presence in calf thymocytes of a GTP-binding protein (G-protein) composed of three polypeptides, 54, 41, and 27 kDa, which was physically and functionally associated with a soluble phosphoinositides-specific phospholipase C (PI-phospholipase C). The properties of this G protein were further investigated with the following results. 1) In addition to the ability to bind [35S]guanosine-5'-[gamma-thio]triphosphate (GTP gamma S), the G-protein exhibited GTPase activity, which was enhanced by Mg2+, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but inhibited by sodium cholate, GTP gamma S and F-.2) The 54-kDa polypeptide was ADP-ribosylated by pertussis toxin and also by endogenous membrane-bound ADP-ribosyltransferase, but none of these three polypeptides was ADP-ribosylated by cholera toxin. 3) The G-protein did not cross-react with either anti-rat brain alpha 1 (alpha-subunit of inhibitory G-protein, G1), alpha 0 (alpha-subunit of other G1-like G-protein, G0) or beta gamma antibodies. 4) Incubation of this G Protein with GTP gamma S caused dissociation of the three polypeptides. 5) The 27 kDa polypeptide showed GTP-binding activity and enhanced the phosphatidylinositol 4,5-bisphosphate hydrolysis by purified PI-phospholipase C. These results suggest that the PI-phospholipase C-associated G-protein in calf thymocytes may be a novel one and that it is involved in the regulation of PI-phospholipase C activity.

Adenosine Diphosphate Ribose↗
PubMed1988

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin.

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.

Animals↗
PubMed1988

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes.

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages.

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.

Animals↗