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K Oguma

Publications and source records attributed to K Oguma.

At least 145 records · Page 8Linked to original sources
PubMed1986

Activation of Clostridium botulinum type E toxin purified by two different procedures.

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.

Botulinum Toxins↗
PubMed1986

Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives.

The biochemical properties of 11 toxigenic and 10 nontoxigenic type C and D strains of Clostridium botulinum were studied. All of the strains examined were motile and hemolytic and produced lipase and liquid gelatin. Fermentation of several sugars and the production of lecithinase, indole, and hydrogen sulfide varied with the strain. The strains were classified into four groups based on their sugar fermentation profiles. The resulting classification was identical to the classification which had been proposed from the relationship between toxin production and phages and similar to the grouping based on the nature of toxin antigenic structures. Lecithinase production was negative in the cells belonging to group III, and indole and hydrogen sulfide production was negative in the cells of groups III and IV. Strains belonging to groups III and IV have many characteristics different from those of groups I and II.

Bacteriophages↗
PubMed1985

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin.

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Animals↗
PubMed1984

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes.

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.

Amino Acids↗
PubMed1984

Comparison of antigenicity of toxins produced by Clostridium botulinum type C and D strains.

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.

Animals↗
PubMed1984

Analysis of antigenicity of Clostridium botulinum type C1 and D toxins by polyclonal and monoclonal antibodies.

Clostridium botulinum type C1 toxin was purified from C-Stockholm (C-ST), and D toxin was purified from D-1873 and D-South African. Polyclonal antibodies against these toxins were prepared in rabbits. Twenty-eight monoclonal antibodies to these toxins were also prepared with BALB/c myeloma cells. The antibodies were analyzed by both enzyme-linked immunosorbent assay (ELISA) and a toxin neutralization test. ELISA was performed with the three purified toxins and heavy-chain (Hc) and light-chain (Lc) components derived from C-ST and D-1873 toxins. A neutralization test was carried out with 11 toxin preparations (7 from type C and 4 from type D cultures). ELISA results indicated that there exists at least one common antigenic determinant on each of the Hc and Lc components of the three purified toxins. The results of the neutralization test also indicated that type C1 and D toxin preparations contain several common antigenic sites in their molecules. Some are common to toxins from several specific cultures, whereas others are common to toxins from a large number of cultures. It was speculated that toxins from two type C strains are composed of Hc and Lc components which are somewhat similar to those of D-1873 and C-ST toxins, respectively.

Animals↗
PubMed1984

Immunopharmacologic profiles of a thiol protease inhibitor, L-trans-dicyclohexyl epoxysuccinate.

Ep-1, L-trans-Dicyclohexyl epoxysuccinate, is a synthetic and specific inhibitor of thiol proteases. The effects of this inhibitor on some immunological parameters were examined in normal and immunity-impaired mice and rats. In the cultures of splenocytes obtained from the mice treated with Ep-1, it enhanced the lymphocyte blast transformation induced by both suboptimal and optimal concentrations of concanavalin A (Con A) and Lens culinaris (LC). The in vivo administration of Ep-1 caused a depression of the plaque forming cells (PFC) for sheep red blood cell (SRBC) and enhanced the delayed-type hypersensitivity (DTH) for bovine serum albumin (BSA) as well as mixed lymphocytes cultures (MLC). Furthermore, Ep-1 demonstrated a preventive effect on adjuvant arthritic rats. The relevance of immunological regulation and the mode of action of Ep-1 as a thiol protease inhibitor are discussed in these findings.

Adjuvants, Immunologic↗
PubMed1983

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes.

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.

Animals↗
PubMed1982

Four different monoclonal antibodies against type C1 toxin of Clostridium botulinum.

Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid. About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA). Four different hybridoma cell lines, no. 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies. The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests. Three monoclonal antibodies, no. 9, 14, and 17, reacted with C-ST toxin, but only no. 17 highly neutralized the toxin. These antibodies did not react with type D toxins. On the contrary, no. 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins. This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction. The neutralization profiles of C-ST toxin by no. 12 and 17 antibodies were different in a time-to-death test of mice. The mechanisms of neutralization by no. 12 and 17 may be different.

Animals↗
PubMed1981

Papain inhibitions by optically active E-64 analogs.

Modification of E-64 focused on the terminal agmatine for practical use led to some potent analogs which were successfully obtained by a stereoselective synthesis using D-tartaric acid as a starting material. Ep-475 (id. E-64-c), in which the agmatine was replaced by 3-methyl-butylamine, was found to react with the essential SH of papain in accord with the decrease of activity. [3H]Ep-475 was irreversibly incorporated into papain in an equimolar ratio. None of the analogs showed any effect on thiol enzymes other than proteolytic ones.

Agmatine↗
PubMed1981

Homogeneity and heterogeneity of toxins produced by Clostridium botulinum type C and D strains.

Five Clostridium botulinum strains were used in the present work, two of type C, C-Stockholm (C-ST) and C-CB19, and three of type D, D-South African (D-SA), D-1873, and D-CB16. The toxins, except for those of C-CB19 and D-CB16, were purified, and antisera were prepared in rabbits. To clarify the antigenicity of the toxins, neutralization and agar gel double-diffusion tests were performed. Anti-C-ST toxin serum neutralized two kinds of type C(1) toxin to a similar extent. Antisera against D-SA and D-1873 toxins, however, showed different neutralizing activity toward three type D toxins. Precipitin lines formed between D-SA and D-1873 toxins, and their antisera spurred to each other. From anti-D-SA toxin serum, two neutralizing fractions, one which neutralized D-SA, D-1873, and D-CB16 and one which neutralized only D-SA, were obtained. These results indicate that the antigenicities of D-SA and D-1873 toxins are not identical. Anti-C-ST toxin serum produced cross-neutralization on type D toxins, although the neutralization titer differed depending on the kind of toxin used. A precipitin line was formed with D-SA toxin, but not with D-1873; the developed line spurred to the C-ST toxin precipitin line. Two anti-D toxin sera also caused cross-neutralization on type C(1) toxins. However, the neutralizing activity of each serum to the same type C(1) toxin was different, and only anti-D-SA toxin serum developed a precipitin line with C-ST toxin which spurred to the D-SA toxin precipitin line. From anti-D-SA toxin serum, two different fractions capable of neutralizing C(1) and D toxins were obtained; one neutralized C-ST, C-CB19, and D-SA toxins, but not D-1873 and D-CB16, and the other neutralized all five toxins. There may be at least two common parts among C-ST, C-CB16, and D-SA toxin molecules.

Antigens, Bacterial↗
PubMed1980

Antigenic similarity of toxins produced by Clostridium botulinum type C and D strains.

Antisera against purified type C1 toxin of Clostridium botulinum and its heavy-chain component cross-neutralized type D toxin. Antisera against partially purified type D toxin cross-neutralized type C1 toxin. From the latter serum, a component which neutralized only type D toxin and a component which equally neutralized both C1 and D toxins were obtained. We concluded that the cross-neutralization was not due to the fact that type C and D strains produce both C1 and D toxins but rather to the fact that the toxins have an antigen(s) common to their molecules. The results of the agar gel-double-diffusion test also supported this conclusion.

Antibody Specificity↗
PubMed1979

High and low toxin production by a non-toxigenic strain of Clostridium botulinum type C following infection with type C phages of different passage history.

Toxin production in Clostridium botulinum types C and D is governed by specific bacteriophages. Prior passages of a phage controlling type C toxin production caused subsequently lysogenized bacteria to become variably toxigenic. This appears to be one of the causes of the decrease in toxigenicity which is common in some type C and D strains. The morphology of bacteria was also changed from rod-shaped to filamentous by infection with a successively propagated phage.

Bacteriophages↗