Biomedical subjects

Tohru Ohyama

Publications and source records attributed to Tohru Ohyama.

24 records · Page 2Linked to original sources

PubMed2003

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia.

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.

Amino Acid Sequence↗

PubMed2002

Spontaneous nicking in the nontoxic-nonhemagglutinin component of the Clostridium botulinum toxin complex.

The nontoxic-nonhemagglutinin (NTNHA) component, in both isolated form and the neurotoxin (NT)/NTNHA complexed form, was prepared protease-free from toxin complexes produced by Clostridium botulinum type D strain 4947. NTNHA in both preparations was found to be spontaneously converted to the nicked NTNHA form leading to 15- and 115-kDa fragments with the excision of several amino acid residues at specific sites on SDS-PAGE during long-term incubation, while that of the NT/NTNHA/hemagglutinin complexed form remained unnicked single-chain polypeptides under the same conditions. Considering that the NTNHA preparation contained small amounts of the nicked form of NTNHA and the addition of trypsin accelerated the cleavage, it is speculated that a nicked form of NTNHA remaining after the purification and/or NTNHA itself catalyzes the cleavage of intact NTNHA.

Amino Acid Sequence↗

PubMed2002

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Administration, Intranasal↗

PubMed2001

In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.

Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.

Amino Acid Sequence↗

PubMed2001

Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA(+)-PTX) binds to oligosaccharides containing Gal beta1-4GlcNAc through one subcomponent of haemagglutinin (HA1).

Haemagglutinin (HA) activity of Clostridium botulinum type A 19S and 16S toxins (HA-positive progenitor toxin; HA(+)-PTX) was characterized. HA titres against human erythrocytes of HA(+)-PTX were inhibited by the addition of lactose, D-galactose, N-acetyl-D-galactosamine and D-fucose to the reaction mixtures. A direct glycolipid binding test demonstrated that type A HA(+)-PTX strongly bound to paragloboside and some neutral glycolipids, but did not bind to gangliosides. Type A HA(+)-PTX also bound to asialoglycoproteins (asialofetuin, neuraminidase-treated transferrin), but not to sialoglycoproteins (fetuin, transferrin). Although glycopeptidase F treatment of asialofetuin abolished the binding of HA(+)-PTX, endo-alpha-N-acetylgalactosaminidase treatment did not. Thus these results can be interpreted as indicating that type A HA(+)-PTX detects and binds to Gal beta 1-4GlcNAc in paragloboside and the N-linked oligosaccharides of glycoproteins. Regardless of neuraminidase treatment, type A HA(+)-PTX bound to glycophorin A which is a major sialoglycoprotein on the surface of erythrocytes. Both native glycophorin A and neuraminidase-treated glycophorin A inhibited the binding of erythrocytes to type A HA(+)-PTX. Since the N:-linked oligosaccharide of glycophorin A is di-branched and more than 50% of this sugar chain is monosialylated, type A HA(+)-PTX probably bound to the unsialylated branch of the N-linked oligosaccharide of glycophorin A and agglutinated erythrocytes. One subcomponent of HA, designated HA1, did not agglutinate native erythrocytes, although it did bind to erythrocytes, paragloboside and asialoglycoproteins in a manner quite similar to that of HA(+)-PTX. These results indicate that type A HA(+)-PTX binds to oligosaccharides through HA1.

Acetylgalactosamine↗

PubMed1999

Characterization of haemagglutinin activity of Clostridium botulinum type C and D 16S toxins, and one subcomponent of haemagglutinin (HA1).

The 16S toxin and one subcomponent of haemagglutinin (HA), designated HA1, were purified from a type D culture of Clostridium botulinum by a newly established procedure, and their HA activities as well as that of purified type C 16S toxin were characterized. SDS-PAGE analysis indicated that the free HA1 forms a polymer with a molecular mass of approximately 200 kDa. Type C and D 16S toxins agglutinated human erythrocytes in the same manner. Their HA titres were dramatically reduced by employing erythrocytes that had been previously treated with neuraminidase, papain or proteinase K, and were inhibited by the addition of N-acetylneuraminic acid to the reaction mixtures. In a direct-binding test to glycolipids such as SPG (NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc beta1-Cer) and GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-Cer), and glycoproteins such as glycophorin A and/or B prepared from the erythrocytes, both toxins bound to sialylglycolipids and sialoglycoproteins, but bound to neither neutral glycolipids nor asialoglycoproteins. On the basis of these results, it was concluded that type C and D 165 toxins bind to erythrocytes through N-acetylneuraminic acid. HA1 showed no haemagglutination activity, although it did bind to sialylglycolipids. We therefore speculate that binding to glycoproteins rather than to glycolipids may be important in causing haemagglutination by type C and D 16S toxins.

Bacterial Toxins↗