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Biomedical subjects

G Sakaguchi

Publications and source records attributed to G Sakaguchi.

At least 55 records · Page 3Linked to original sources
PubMed1990

Assay in mice for low levels of Clostridium botulinum toxin.

When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LD50 was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed. If this symptom is taken as a marker, 1.0 mouse intraperitoneal LD50 can be detected within 6 h and 0.1 LD50 within 24 h. The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity. Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LD50, a linear relationship was obtained between the log dose and the score. By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LD50 can be titrated accurately and easily.

Abdominal Muscles↗
PubMed1990

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule.

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.

Antibodies, Bacterial↗
PubMed1990

Titration of botulinum antitoxin of low levels by the score method.

Botulinum antitoxin is commonly titrated by injecting a mixture of toxin and antitoxin into mice and by utilizing deaths as a marker to measure the amount of unneutralized toxin. We attempted to titrate antitoxin by converting the severity of symptoms (notably palsy) and time-to-death in days into scores. In neutralization tests with toxin levels at 5.9 LD50 and 23.5 LD50, a linear relationship was obtained for antitoxin dose in a range between 0.03 to 0.003 IU/ml. Statistical analysis showed that homogeneity of variance or slope was not denied for the scores obtained on any day from the first to the fourth days after injection, demonstrating that this method can titrate accurately antitoxin of such a low level as 0.003 IU/ml within 4 days after injection.

Animals↗
PubMed1990

Incidence of Clostridium botulinum in honey of various origins.

By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one. Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C. Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F). The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%). It was estimated that most positive samples contained spores in one per gram or lower concentrations. One sample contained 4 type A spores per gram and another 36-60 type F spores per gram. No distinct biochemical properties were found with the honey isolates.

Clostridium botulinum↗
PubMed1990

Comparative studies on Clostridium botulinum type A strains associated with infant botulism in Japan and in California, USA.

Twenty strains of Clostridium botulinum type A associated with infant botulism cases, six in Japan and 14 in California, USA, were compared in their characters. All six Japanese strains produced medium-sized progenitor toxin (M toxin; Mr 300 k) but no hemagglutinin and showed lower 50% infective doses (ID50) in the infant mouse test; whereas most American strains produced large-sized progenitor toxin (L + LL toxins; Mr 500 k and 900 k) and hemagglutinin in addition to M toxin and showed higher ID50 in infant mice. No marked difference in the biochemical properties was found between the two groups except for two American strains.

Animals↗
PubMed1990

Attempts to quantity Clostridium botulinum type A toxin and antitoxin in serum of two cases of infant botulism in Japan.

Serum samples taken from two infant botulism cases during hospitalization were titrated for botulinum toxin by both the intraperitoneal (ip) injection method and the score method in mice. By the ip method, in which death is the only parameter, such low levels of toxin as lower than 4 ip LD50/ml may not be titrated even though the surviving mice show abdominal palsy. By the score method based on the degree of abdominal palsy, such low levels of toxin as 1.1 and 0.8 ip LD50/ml were detected in specimens of one of the patient's serum. No antitoxin was demonstrated in either case of infant botulism by applying the score method. It is not known whether spontaneous recovery from infant botulism is due to the antitoxin production.

Botulinum Antitoxin↗
PubMed1989

[Infant botulism was confirmed in Ehime Prefecture].

The third case of infant botulism in Japan is reported. A four-month-old baby boy suddenly had weakness of suckling force, constipation and generalized hypotonicity. He was the product of normal gestation, labor and delivery. Growth and development were normal until he was nineteen weeks old. He received fruit-juice and honey daily. By bacteriological examination, Clostridium botulism type A was isolated from his feces and the honey which he had received. Type A toxin was detected from his feces but not from his serum.

Botulism↗
PubMed1989

Immunological characterization of papain-induced fragments of Clostridium botulinum type A neurotoxin and interaction of the fragments with brain synaptosomes.

After treatment of Clostridium botulinum type A neurotoxin with papain, three fragments (Mrs, 101,000, 45,000, and 43,000) were purified by hydrophobic and ion-exchange chromatography with a high-performance liquid chromatographic system. Immunoblotting analyses with monoclonal antibodies showed that the 101,000-dalton fragment consisted of the light chain and a part of the heavy chain (H-1 fragment) linked together by a disulfide bond, and the other two fragments were correlated to the remaining portion of the heavy chain (H-2 fragment). The 45,000- and 43,000-dalton fragments effectively competed for binding of the 125I-labeled neurotoxin to synaptosomes, while no inhibition was observed with the 101,000-dalton fragment. The results indicate that the H-2 fragment interacts with the binding site on the neural membrane. The binding of the neurotoxin was impaired by treatment of synaptosomes with neuraminidase. Incorporation of gangliosides into neuraminidase-treated synaptosomes resulted in the restoration of binding. The results suggest that gangliosides are one of the components of the toxin-binding site.

Animals↗
PubMed1989

Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.

Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000- and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.

Aeromonas↗
PubMed1989

Association between the cell-wall peptidoglycan and the progenitor toxin of Clostridium botulinum type C.

Clostridium botulinum type C progenitor toxin with a molecular weight of 500 k daltons (C1 L toxin), purified from the bacterial cells, bound at pH 2 to the cell-wall peptidoglycan derived from certain strains. The carbohydrate moiety of the peptidoglycan contained arabinose and galactose at a certain ratio, both of which may directly be associated with the binding. The binding, being dependent on the quality and quantity of the sugars, enhanced the oral toxicity of the toxin to the chicken as well as the mouse.

Animals↗
PubMed1989

Implication of coprophagy in pathogenesis of chicken botulism.

Oral administration of 1 x 10(7) viable spores of Clostridium botulinum type C killed the chickens kept on a board floor to allow them coprophagy, whereas the same dose of the spores failed to develop symptoms in those kept on a wire-net floor not to allow them coprophagy. Type C toxin was detected in the cecal droppings of the chickens of both the groups after feeding the spores and also in serum of symptomatic as well as asymptomatic chickens kept on a board floor. Thus, coprophagy, by which chickens ingest type C toxin (C1 L toxin) and the bacterial cells, seems to be a prerequisite for development of chicken botulism.

Animals↗
PubMed1988

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction.

Zn2+ (10-100 microM) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca2+. Botulinum type A toxin (BTXA, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn2+ (100 microM) restored MEPPs and increased their frequency after they had been abolished by BTXA in Ca2+ -free solutions. The antagonistic effect of Zn2+ in the Ca2+ -free solution was reduced by exposing the diaphragm to the toxin in the Ca2+ -free solutions containing high K+. Thus, the action of BTXA is probably enhanced by depolarization of the motor nerve terminals.

Animals↗
PubMed1988

Purification of Clostridium botulinum type G progenitor toxin.

Clostridium botulinum type G cultured for 6 days at 30 degrees C in proteose peptone-yeast extract-glucose medium produced toxin of 1.3 x 10(4) LD50/ml. The toxin was precipitated at pH 4.0, extracted with 0.2 M phosphate buffer, pH 6.0, and activated with trypsin. Sonic treatment and trypsinization of the residual precipitate released additional toxin, the toxicity of which corresponded to that detected in whole culture. Activated toxin obtained from the first extract and that from the residual precipitate were combined and purified by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second gel filtration on Sephadex G-200. The yield of purified toxin from 10 liters of culture was 22.9 mg an 1.1 X 10(8) mouse ip LD50 with a specific toxicity of 3.0 X 10(7) mouse ip LD50/mg nitrogen. The molecular weight of the toxin was about 500,000, corresponding to that of L toxin of the other types. No M nor LL toxin was detected.

Botulinum Toxins↗
PubMed1988

Production of monoclonal antibody against Aeromonas hydrophila haemolysin.

Two hybridoma cell lines that produce monoclonal antibodies against Aeromonas hydrophila haemolysin were established by fusion of myeloma and spleen cells obtained from a mouse immunised with haemolysin detoxified with tetranitromethane. Enzyme-linked immunosorbent assay (ELISA) showed that the two purified monoclonal antibodies, B7 and B11, recognised the same epitope on the haemolysin molecule. Antibody B7 neutralised the haemolytic and enterotoxic activities of the haemolysin. It is concluded that the same site on the haemolysin molecule is responsible for both haemolytic and enterotoxic activities.

Aeromonas↗