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Biomedical subjects

S Toyoshima

Publications and source records attributed to S Toyoshima.

At least 109 records · Page 6Linked to original sources
PubMed1989

N-(cyclohexylcarbonyl)-D-phenylalanines and related compounds. A new class of oral hypoglycemic agents. 2.

A series of analogues of N-(cyclohexylcarbonyl)-D-phenylalanine (5) have been synthesized and evaluated for their hypoglycemic activity. Relationships were studied between the activity and the three-dimensional structure of the acyl moiety, which was characterized by high-resolution 1H NMR spectroscopy and MNDO calculations. The role of the carboxyl group of the phenylalanine moiety was also studied by comparing the activities of the enantiomers, the decarboxyl derivative, the esters, and the amides of the phenylalanine derivatives. Thus, the structural requirements for possessing hypoglycemic activity was elucidated and a highly active compound, N-[(trans-4-isopropylcyclohexyl)carbonyl]-D-phenylalanine (13) was obtained, which showed a 20% blood glucose decrease at an oral dose of 1.6 mg/kg in fasted normal mice.

Administration, Oral↗
PubMed1989

Concanavalin A receptor(s) possibly interacts with at least two kinds of GTP-binding proteins in murine thymocytes.

A part of the GTP gamma S-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as Gi (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti-alpha i (alpha subunit of Gi) immunoblotting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP gamma S-binding activity was detected in the Con A-bound fraction, suggesting that Gi physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP gamma S-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phosphatidylinositol 4,5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin E1 and isoproterenol-induced increases of cellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes.

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.

Amino Acid Sequence↗
PubMed1989

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity.

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.

Animals↗
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

[Trends of the patients with malocclusion on dental clinic of pediatric dentistry].

The factors which determine the situation of continuation in malocclusion treatment and characteristic of patient with malocclusion in pediatric dentistry were estimated from the status and the trend of patients undergoing denture guidance at the outpatient clinic for pediatric dentistry. The following results were obtained. 1. Among the patients undergoing denture guidance, a majority were females. Among the different types of malocclusion, the reversed occlusion was the most frequent, being 54.3% among all malocclusions. 2. In particular the age for first visit was from under 3 years of age with reversed occlusion, and at this age, females were more numerous than males. 3. The first examination for denture guidance was related to the time which made effective diagnosis possible, and the patients with reversed occlusion had their first examinations consistently from 3 years of age, and the patients with crowding had their first examinations from 6 years of age to 11 years of age. 4. From the difference between the age of first visit and first examination, it was shown that the patients with reversed occlusion did not have their first examination under 3 years of age and for the patients with crowding who visited first at 3, 4 and 5 years of age, the first examination was performed after 6 years of age. The patients with crowding in the group who came for consultation by reference were later about two years compared with other two groups in the difference of motivation for visiting. 5. It is suggested that the factors which had an influence upon the situation of continuation of malocclusion treatment were; an existence of the use of an appliance, a substantial program of correspondence in regard to dental care for malocclusion before malocclusion treatment and concrete indication of the treatment.

Child↗
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

N-acylphenylalanines and related compounds. A new class of oral hypoglycemic agents.

N-Benzoyl-DL-phenylalanine (1) was found to possess hypoglycemic activity. A series of the analogues of compound 1 were prepared and evaluated for their blood glucose lowering activity. Both the steric effects of the phenylalanine moiety and the effects of variations in the acyl moiety were investigated. This study elucidated some of the structure-activity relationships and led to the development of N-(4-ethylbenzoyl)-D-phenylalanine (34), which was 50 times more potent than the initial compound 1.

Animals↗
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

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↗
PubMed1988

Adenomyoepithelioma of the breast. A case report with an immunohistochemical study.

We treated a 62-year-old woman with adenomyoepithelioma of the breast, an extremely rare tumor, which showed a bicellular pattern of ductal and myoepithelial elements bearing some histologic resemblance to pleomorphic adenoma of the salivary gland. The tumor was made up of cells positive for actin and S-100 protein, and some positive for epithelial membrane antigen (EMA). Thus, this tumor showed biphasic differentiation towards myoepithelial cells and to duct epithelial cells. Compared with findings in related tumors reported in the literature, the myoepithelial cells in the present tumor were less frequently spindle-shaped and had abundant eosinophilic or clear cytoplasm. A simple mastectomy was done and at the time of writing 20 months later there has been no recurrence. Adjuvant chemotherapy was not prescribed.

Actins↗
PubMed1987

Sebaceous gland tumor of the ovary.

A sebaceous gland tumor of the ovary was detected in a 60-year-old woman who underwent right salpingo-oophorectomy for a right ovarian cyst. The cyst was unilocular, weighed 820 g, and was filled with sebaceous material containing a few hair shafts. There was a protruded mass composed of lobules of mature or immature sebaceous cells over the inner surface of the cyst wall. She has been well for 4 years and 2 months after the surgery. This is the second well-documented case of this extremely rare type of tumor. This lesion is teratogenic with unilateral development of the sebaceous glands and malignant characteristics are nil.

Diagnosis, Differential↗
PubMed1987

Protein-O-carboxylmethyltransferase from cytosol and membranes of chicken erythrocytes.

Cytosolic protein-O-carboxylmethyltransferase was purified more than 4,000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane-associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1 microM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71 microM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.

Animals↗
PubMed1987

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein.

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.

Animals↗
PubMed1986

Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP.

The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.

Adenosine Triphosphate↗
PubMed1986

Phosphatidylinositol-specific phospholipase C of murine lymphocytes.

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.

Animals↗