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Biomedical subjects

M Beppu

Publications and source records attributed to M Beppu.

36 records · Page 2Linked to original sources
PubMed1987

Detection of oxidized lipid-modified erythrocyte membrane proteins by radiolabeling with tritiated borohydride.

Human erythrocyte ghosts treated with tert-butyl hydroperoxide or ADP-Fe3+ incorporated radioactivity on reduction with tritiated borohydride. The tritium incorporation closely correlated with membrane lipid oxidation as assessed by the formation of thiobarbituric acid-reactive substances and fluorescent substances. Treatment of ghosts with the inducers in the presence of butylated hydroxytoluene, thiourea, or desferrioxamine suppressed the tritium incorporation in the subsequent reduction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tritiated ghost proteins showed that the label was incorporated into the intermolecularly cross-linked and the uncross-linked proteins of bands 1, 2, 3, 4.1, 4.2, 5 and 6, and into the noncross-linked glycophorin A (PAS-1). Glycophorin A was hardly cross-linkable but modified during membrane lipid oxidation. Possible candidates for producing borohydride-reducible functions in the proteins are various mono- and bifunctional aldehydes, as well as those for producing fluorescence and cross-links. A part of thiobarbituric acid-reactive or fluorescent substances may be involved in borohydride reduction and tritium labeling.

Aldehydes↗
PubMed1987

Periodate-induced lipid oxidation of erythrocyte membranes.

Exposure of human erythrocyte ghosts to 0.2-5 mM periodate at 0 C for 15 min resulted in an increase of thiobarbituric acid-reactive substances and fluorescent materials in the membrane. This increase was suppressed by radical scavengers, butylated hydroxytoluene and thiourea, indicating that periodate caused lipid oxidation of ghosts. A role of hemoglobin in the periodate-induced lipid oxidation of ghosts was suggested by the fact that the oxidation was augmented by hemoglobin and inhibited by an iron chelator, desferrioxamine. Treatment of ghosts with periodate caused membrane protein cross-linking with and without disulfide bridge. Erythrocytes were also susceptible to lipid oxidation by periodate, but only disulfide-mediated protein cross-linking was observed. Erythrocytes treated with neuraminidase or trypsin were less susceptible, but those treated with neuraminidase along with galactose oxidase were nearly as susceptible as untreated cells. The effect of galactose oxidase was diminished by reduction of the enzyme-treated cells with borohydride. These results indicate that the aldehyde moieties generated by periodate at the sialyl residues or those generated by galactose oxidase at the terminal galactosyl or N-acetyl galactosaminyl residues of the membrane glycoconjugates play a stimulating role in the periodate-induced membrane lipid oxidation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Epiglycanin-immunoreactive glycoproteins in mouse fetal tissues and fetal cells in culture.

Fetal tissues from time-pregnant female A/J mice of 16- and 19-day pregnancies and from neonates 1 day after birth, as well as from fetal cells in culture, absorbed significant amounts of anti-epiglycanin antibody. Detergent-solubilized glycoproteins, with epiglycanin activity, from fetal tissues and cells were separated by polyacrylamide gel electrophoresis, and the protein bands electroblotted onto a nitrocellulose gel. After the antigens were labeled with rabbit anti-epiglycanin antiserum and [125I]epiglycanin, autoradiography revealed two major bands containing the antigenic determinant at Mr 90,000 and 82,000. Bands of similar molecular weights, but with no demonstrated immunologic cross-reactivity, were observed by fluorography, if intact cells prior to solubilization were labeled by galactose oxidase followed by sodium borotritiide. Immunoreactive epiglycanin activity could be destroyed by Pronase, endo-N-acetyl-alpha-D-galactosaminidase (Diplococcus pneumoniae), or periodate oxidation. Activity was enhanced with neuraminidase. The spleen, liver, or erythrocytes from adult A/J mice did not possess the antigen, but incubation of adult spleen or liver with neuraminidase (Vibrio cholerae) exposed the epitope.

Animals↗
PubMed1979

Preparation of concanavalin A-ricin A-chain conjugate and its biologic activity against various cultured cells.

For the development of therapeutic agents that possess tissue-specific carriers, a method was devised to synthesize an artificial protein hybrid conjugate containing a moiety which binds to a cell membrane receptor and an active fragment of a toxic protein. By the introduction of an activated sulfhydryl group into concanavalin A (Con A), a conjugate of Con A and the ricin A-chain cross-linked with a disulfide linkage was synthesized. The purified conjugate was studied with regard to its inhibitory activity against protein synthesis in cell-free and cultured cell systems. The Con A-rich A-chain conjugate retained about one-third the inhibitory activity of ricin in a cell-free protein synthesis system. It also was highly toxic to cultured normal cells. These results indicate that the conjugate is a structural and functional analog of ricin and that the original membrane-binding chain (B-chain of ricin) could be replaced by Con A. Transformed cells were insensitive to this conjugate and required a longer preincubation time. The sensitivity of the normal cells was reduced in the presence of local anesthetics.

Animals↗
PubMed1979

Covalently cross-linked monovalent, divalent, and tetravalent derivatives of concanavalin A.

Dimeric succinyl-concanavalin A was cross-linked with ethylenediamine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide a condensing agent. Thus, a divalent dimer and a tetravalent tetramer composed mostly of covalently cross-linked subunits bearing altered net charges were obtained. Photoaffinity labeling of the cross-linked dimer with p-azidophenyl alpha-D-mannopyranoside resulted in a specific label for its saccharide-binding site and yielded a nonvalent dimer and a monovalent dimer (showing no subunit exchange). However, hemagglutination and glycogen precipitation data suggested that the labeled binding site is shielded but not destroyed by the label and can still bind weakly an external saccharide ligand possibly due to unsteadiness of the shielding label. Although nonvalent and monovalent derivatives were mitogenic as well as divalent and tetravalent derivatives for mouse splenic lymphocytes, binding and stimulation experiments indicated that their stimulating efficiencies after binding to the cells were far lower than those of the multivalent counterparts. Their activities were inhibited by methyl alpha-D-mannopyranoside, suggesting that the weak activities of nonvalent and monovalent derivatives were due to the labeled sites entirely and partly, respectively. We suggest that the triggering of lymphocyte mitogenesis by concanavalin A may depend on cross-linkage of cell surface receptors.

Animals↗
PubMed1979

[An experimental study on the effects of the plating method for bone fractures--with reference to bone union and bone strength (author's transl)].

The plating method for bone fractures is in wide use. The author studied both new bone formation and bone atrophy of the cortex of fracture site after long periods of plating. This study is concerned with the bone strength of the fracture site as influenced by rigid plate fixation, and also with bone union after the removal of the plate. The experimental results, using mature rabbits as subjects, showed the following: X-rays of the bone union at the fracture site treated by the plating method confirmed evident union at 8 weeks after surgery. The bone strength at the region of this union, as tested by the bending method (an Instron Type Testing Machine was used), showed its highest values at 28 weeks after surgery. Yet, this was only 60% of the bone strength as measured at a non-fracture site; it then gradually declined in strength. On the other hand, in the case where the plate had been removed 8 weeks after surgery, bone strength was recovered to the same level as that of non-fracture sites 16 weeks after surgery. In conclusion, rigid plate fixation for a long period may result not only in progressive bone atrophy of the cortex but also in a delayed remodeling of the bone at the region of the bone union. It is, therefore, very important to remove the plate as early as possible once union has been achieved. Careful observation, however, is necessary during the short time between plate removal and the period when bone strength becomes again normal.

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↗
PubMed1975

Photoaffinity labeling of concanavalin A. Preparation of a concanavalin A derivative with reduced valence.

Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A.

Affinity Labels↗
PubMedDate not supplied

Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins.

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.

Animals↗