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Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 469 records · Page 26Linked to original sources
PubMed1976

Purification and biological activities of pokeweek (Phytolacca americana) mitogens.

Five mitogens, designated Pa-1 through Pa-5, were purified from the roots of pokeweed (Phytolacca americana) by means of ethanol fractionation, DEAE-cellulose column chromatography, affinity chromatography on a column of desialized human erythrocyte glycopeptide-Sepharose 4B, and gel filtration. Among these mitogens, only Pa-1 was mitogenic for both murine B-cells and T-cells, and the other mitogens were T-cell mitogens. Binding experiments with 125I-labeled Pa-1, a potent mitogen for B-cells, and with 125I-labeled Pa-2, the strongest T-cell mitogen, revealed that murine B-cells have more receptor sites for Pa-1 than for Pa-2 and murine T-cells have more receptor sites for Pa-2 than for Pa-1. The change of membrane fluidity within 30 min after binding of the mitogens to murine B- and T-cells was measured by fluorescence polarization of fluorescent hydrocarbon, 1,6-diphenyl-1,3,5-hexatriene, embedded in the membrane. Pa-1 induced increase of membrane fluidity of B-cells more markedly than Pa-2, whereas Pa-2 had a larger effect on the membrane fluidity of T-cells than Pa-1. Although both Pa-1 and Pa-2 stimulated the incorporation of 32 Pi into phosphatidylinositol of murine T-cells, neither Pa-1 nor bacterial lypopolysaccharide induced the activation of phospholipid metabolism of murine B-cells.

Animals↗
PubMed1976

Synthesis of O-beta-D-galactopyranosyl-(1 linked to 4)-O-(2acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 linked to 2)-D-mannose and its interaction with various lectins.

2-Methyl-[3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha D-glucopyrano]-[2,1-d]-2oxazoline (4) was prepared from 2-acetamido-3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyranosyl chloride. Condensation of 3,4:5,6-di-O-isopropylidene-D-mannose dimethyl acetal with 4 in the presence of a catalytic amount of p-toluenesulfonic acid afforded O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1 linked to 4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-(1 linked to 2)-3,4:5,6-di-O-ispropylidene-D-mannose dimethyl acetal (6) in 8.6% yield. Catalytic deacetylation of 6 with sodium methoxide, followed by hydrolysis with dilute sulfuric acid, gave O-beta-D-galactopyranosyl-(1 linked to 4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 linked to 2)-D-mannose (7). The inhibitory activities of 7 and related sugars against the henagglutinating activities of various lectins were assayed, and 7 was found to be a good inhibitor against Phaseolus vulgaris hemagglutinin.

Acetylglucosamine↗
PubMed1976

Activation of mouse splenic lymphocyte guanylate cyclase by calcium ion.

The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter.

Animals↗
PubMed1976

Preparation of monovalent succinyl-concanavalin A and its mitogenic activity.

Monovalent succinyl-concanavalin A was prepared by photoaffinity labeling of succinyl-concanavalin A with p-azidophenyl a-D-mannopyranoside. This concanavalin A derivate showed a weak but definite mitogenic acitivity against mouse splenic lymphocytes and human peripheral lymphocytes, suggesting that direct receptor cross-linkage on the cell surface is not an obligatory prerequisite for the activation of lymphocytes.

Affinity Labels↗
PubMed1976

Purification and characterization of beta-N-acetylhexosaminidases and beta-galactosidase from Streptococcus 6646 K.

Three beta-N-acetylhexosaminidases [EC 3.2.1.52] and one beta-galactosidase [EC 3.2.1.23] were purified from the culture filtrate of streptococcus 6646 group K by a combination of column chromatographies on p-aminophenyl beta-D-thiogalactopyranoside-substituted Sepharose and N-(paminophenyl)oxamic acid-substituted Sepharose. These beta-N-acetylhexosaminidases showed optimal activities between pH 5.0 and 5.5 and could hydrolyze synthetic and glycopeptidic substrates. Glycolipids such as GM2, asialo-GM2, and globoside I were no susceptible to these beta-hexosaminidases. beta-Galactosidase, which was purified more than 11,000-fold, had a substrate specificity rather similar to that of beta-galactosidase from E. coli. This enzyme was inhibited by EDTA and activated by Mn2+, Ca2+, and Mg2+. Problems pertinent to the application of affinity chromatography to the purification of glycosidases are also discussed.

Cations, Divalent↗
PubMed1976

Studies on the hydrazinolysis of glycoproteins. Core structures of oligosaccharides obtained from Porcine thyroglobulin and pineapple stem bromelain.

Hydrazinolysis of porcine thyroglobulin glycopeptides and of pineapple stem bromelain [EC 3.4.22.4] permitted the isolation of almost intact carbohydrate chains of these glycoproteins. On the basis of permethylation analyses of the released oligosaccharides after reduction with NaBH4, the core structures of Unit A-type and Unit B-type carbohydrate chains of porcine thyroglobulin were deduced to be Manalpha1 leads to 6[Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[Ralpha1 leads to 6]GlcNAc leads to Asn (Unit A-type, R=H; Unit B-type, R=Fuc), and that of bromelain was found to be Manalpha1 leads to 6[R'1 leads to 2]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[R1 leads to 3]GlcNAc leads to Asn (R'=Xylbeta and R=Fucalpha, or R'=Fucalpha and R=Xylbeta). From these results, it appears that the hydrazinolysis method is applicable to wide variety of glycoproteins which have an N-glycosylamine linkage between the carbohydrate and peptide moieties, regardless of the type of linkage to the most proximal N-acetylglucosamine residue which is bound to asparagine.

Animals↗
PubMed1976

Acute anticonvulsant effects of diphenylhydantoin, phenobarbital, and carbamazepine: a combined electroclinical and serum level study in amygdaloid kindled cats and baboons.

This preliminary study utilizing the kindling model of established epilepsy has provided information about the most effective routes of administration for diphenylhydantoin, phenobarbital, and carbamazepine in baboons and cats. The time of peak plasma levels for these drugs has been demonstrated in these animals so that experimental protocols can be designed to deliver the agents at appropriate times prior to the kindling stimulation. In addition, dose-effectiveness data is presented for these species. In 1 baboon, phenobarbital was also seen to suppress photosensitive seizures. The potential usefulness of the kindling model in anticonvulsant research is suggested.

Amygdala↗
PubMed1976

Spontaneous recurrent seizure state induced by daily electric amygdaloid stimulation in Senegalese baboons (Papio papio).

Daily electrical stimulation of the amygdala in Senegalese baboons (Papio papio) resulted in the development of generalized convulsive seizures focal onset through five distinct clinical stages in an average of 72 days. The chronologic pattern of electroclinical features suggested that vertical intrahemispheric ictal dissemination was of primary importance in the progressive seizure development. Some animals developed spontaneous recurrence of both partial complex and primary generalized seizures. The kindling preparation in P. papio represents a unique model of human epilepsy with its secondary generalized convulsive seizure development, spontaneously recurrent partial and primary generalized seizures in the background of predisposed epileptogenic susceptibility.

Amygdala↗
PubMed1976

Cytotoxic activity of purified guinea pig lymphotoxin against various cell lines.

Cytotoxic activity of the purified guinea pig lymphotoxin (GLT) was tested on various blood cells and various cultured cell lines. GLT was generally ineffective on erythrocytes and lymphocytes, but it was somewhat effective on peritoneal exudate cells indicating GLT might play an important role in the regulatory mechanism of the cellular immunity. Considerable difference in target cell sensitivity was observed among the cultured cell lines tested. The sensitivity of L cells to GLT was significantly lowered by the trypsin treatment of the cells. Furthermore, porcine thyroglobulin glycopeptide exerted weak but definite inhibitory activity against GLT. These results suggest that the cytotoxicity of GLT might be triggered by the binding of GLT to the receptor sites of glycoprotein nature on the cell surface.

Animals↗
PubMed1975

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.

Binding Sites↗
PubMed1975

Immunological evidence for the localization of sialoglycosphingolipids at the cell surface of sea urchin spermatozoa.

The localization of sialoglycosphingolipids in the plasma membrane of sea urchin spermatozoa was studied by employing immunological methods including immunolysis of liposomal model membranes. The antibodies produced against these complex lipids were found to agglutinate various sea urchin spermatozoa differently. Both species differences and species similarities in the agglutination were found in spermatozoa of the echinoderm, the sea urchin and the starfish. The agglutination of the sea urchin spermatozoa was inhibited specifically by ceratain carbohydrates. Only a limited number of molecular species of sialoglycosphingolipid were localized at the surface of the plasma membrane of sea urchin spermatozoa cells. Moreover, topographical differences were found in the localization of the sialoglycosphingolipids at the cell surface of spermatozoa.

Agglutination Tests↗
PubMed1975

Lectins from Wistaria floribunda seeds and their effect on membrane fluidity of human peripheral lymphocytes.

Two lectins were isolated from Wistaria floribunda seeds. One is a strong mitogen against human peripheal lymphocytes and has been purified in the previous paper (Toyo-Shima S., Y., Nakano K., Tonomura, A., and Osawa T. (1971) Biochemistry 10, 4457). The other, which is a strong hemagglutinin being devoid of mitogenic activity against normal lymphocytes, has been purified in this paper by affinity chromatography on a Sepharose 6B column followed by DEAE-Sephadex column chromatography. Both lectins were found to be glycoproteins and their molecular weights were estimated to be 136,000 for the equilibrium. The hemagglutinin is composed of four apparently identical subunits of a molecular weight of 35,000 and the mitogen is adimer of 32,000 molecular weight subunit. Binding experiments with 125-I-labeled W. floribunda mitogen revealed that the maximal incorporation of [6-3H]thymidine or 32-PO4 occurred when only 5.2% of the avaliable receptor sites on normal lymphocytes were occupied by the mitogen. Furthermore, the mobility of W. floribunda lectins as well as other lectins bound to the cell receptor sites of normal lymphocytes was determined by fluorescence polarization of fluorescein-labeled lectins. The mitogen lectins tested, have high mobility and Lens culinaris hemagglutinin, have high mobility whereas the nonmitogenic lectins, W. floribunda hemagglutinin, Sophora japonica hemagglutinin, and eel serum anti-H hemagglutin show relatively low mobility. However, W. floribunda hemagglutinin bound to neuraminidase-treated lymphocytes showed relatively high mobility in accord with the fact that this hemagglutinin exerted weak but definite mitogenic activity against neuraminidase-treated lymphocytes. The change of membrane fluidity upon binding of the lectins to normal lymphocytes was also measured by fluorescence polarization of fluorescent hydrocarbon, 1,6-diphenyl-1,3,5-hexatriene, embedded in the membrane. The mitogenic lectins, W. floribunda mitogen and L. culinaris hemagglutinin, increased the membrane fluidity upon binding to lymphocyte cell surface within 30 min, whereas the non-mitogenic lectins, W. floribunda hemagglutinin and S. japonica hemagglutinin, did not effect the membrane fluidity. We suggest that the increase of membrane fluidity is one of the common biochemical events in the earliest stage of lymphocytes transformation.

Binding Sites↗
PubMed1975

Synthesis of 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-D-mannose, and its interaction with D-mannose-specific lectins.

Condensation of 3, 4:5, 6-di-O-isopropylidene-D-mannose dimethyl acetal with 2-methyl-(3, 4, 6-tri-O-acetyl-1, 2-dideoxy-alpha-D-glucopyrano)-[2', 1':4, 5]-2- oxazoline in the presence of a catalytic amount of p-toluenesulfonic acid afforded crystalline 2-O-(2-acetamido-3, 4, 6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3, 4:5, 6-di-O-isopropylidene-D-mannose dimethyl acetal (3) in 25 percent yield. Catalytic deacetylation of 3 with sodium methoxide, followed by hydrolysis with dilute sulfuric acid, gave 2-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-D-mannose (4). Treatment of 3 with boiling 0.5 percent methanolic hydrogen chloride under reflux gave methyl 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-mannopyranoside (5) and methyl 2-o-2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-mannofuranoside (6). The inhibitoryactivities of 4, 5, and 6 against the hemagglutinating and mitogenic activities of Lens culinaris and Pisum sativum lectins and concanavalin A were assayed. From the results of these hapten inhibition studies, subtle differences of specificity between these D-mannose-specific lectins were confirmed.

Binding Sites↗
PubMed1975

Immunochemical and chemical investigations of the structure of glycoprotein fragments obtained from epiglycanin, a glycoprotein at the surface of the TA3-Ha cancer cell.

The structures of the carbohydrate chains present in fragments of a large-molecular-weight glycoprotein, epiglycanin, cleaved from the surface of viable TA3-Ha murine mammary carcinoma ascites cells and purified by gel filtration, were studied by immunochemical and chemical methods. Inhibitory activities for neuraminidase-treated and untreated glycoprotein material in the hemagglutination of NN-specific human erythrocytes by eight purified lectins were determined. Excellent inhibition was obtained in the Bauhinia purpurea, Arachis hypogaea, Iberis amara, and Wistaria floribunda systems, and weak inhibition against the Ricinus communis and Glycine max lectins. No activity against hemagglutination by the Phaseolus vulgaris and Phaseolus limensis lectins was observed. These results, when compared with those obtained by periodate oxidation, alkaline borohydride reduction, and partial methylation, suggest the possible presence of six different carbohydrate chains of 1 to 5 components in length, having as terminal groups N-acetylneuraminic acid, galactose, and 2-acetamido-2-deoxygalactose. All chains are attached to a single polypeptide chain by O-glycosyl bonds involving a 2-acetamido-2-deoxygalactose residue and a serine or threonine residue. It is suggested that the native molecule of epiglycanin of molecular weight 500,000 contains more than 500 carbohydrate chains attached to a single polypeptide chain of similar to 1,300 amino acid units.

Amino Acids↗