Therapeutic use of type F botulinum toxin.
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Aged↗
Biomedical subjects
G Sakaguchi
Publications and source records attributed to G Sakaguchi.
Detection of Clostridium botulinum in natural sweetening.
Various sugar products were examined for contamination with C. botulinum spores. Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey. The heavy contamination of honey with C. botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found. Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa. The fact that some natural sweetenings are contaminated with C. botulinum spores, even in low concentrations, may be food-hygienically important.
Animals↗
Detection of neutral sugars in purified type G botulinum progenitor toxin and the effects of some glycolytic enzymes on its molecular dissociation and oral toxicity.
Arabinose and galactose were detected in purified type G botulinum toxin (Mr about 500,000) of Clostridium argentinense. The i.p. LD50/mg N of type G progenitor toxin was one-tenth, but the oral LD50/mg N twice that of type A-L toxin. The lysozyme-, endo-beta-galactosidase-, and N-glucanase-treated toxins each had a molecular mass of about 300,000. The oral toxicity of the endo-beta-galactosidase or N-glucanase-treated toxin was one-fifth that of untreated progenitor toxin. On DEAE-Sephadex chromatography, the N-glucanase-treated toxin dissociated into two fractions, nontoxic and toxic. SDS-PAGE of the toxic fraction showed a single band with a Mr of about 150,000, and after dithiothreitol treatment, two bands with Mr of 100,000 and 50,000.
Administration, Oral↗
Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin.
Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.
Botulinum Toxins↗
An unusually heavy contamination of honey products by Clostridium botulinum type F and Bacillus alvei.
Many spores (1-60/g) of Clostridium botulinum type F were detected in different containers of honey products of the same brand. Microbiological and physicochemical properties of the contaminated honey were compared with those of the negative one. No difference in pH, hydroxymethyl furfural contents or diastase activity was found between them. The total counts of anaerobes other than C. botulinum and of yeast were also similar, whereas the aerobe counts, which were proportionally related with the C. botulinum counts, were higher in the positive honey than in the negative one. Motile colony-forming Bacillus alvei was predominant among the aerobes. B. alvei stimulated the toxin production by C. botulinum type F in culture medium incubated under aerobic conditions. The high count of C. botulinum in the honey might have been due to the possible stimulation of growth by B. alvei or some other microorganisms at some stage of honey ripening.
Aerobiosis↗
Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties.
Two strains of Clostridium botulinum type A associated with food-borne botulism and six strains associated with infant botulism in Japan were compared in intraintestinal toxin production in infant mice, in vitro toxin and hemagglutinin production, molecular sizes of the toxins, and some other properties. The infant botulism-associated strains, producing M toxin (Mr 300 kDa) but no hemagglutinin, showed significantly lower 50% infective doses in infant mouse intestines. The antigenicities of the toxin differed between the two groups, while the biochemical properties of the cultures did not. Besides infant botulism-associated strains, this set of properties were found only in a strain isolated from honey of South American origin.
Animals↗
Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin.
To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.
Animals↗
Immunological characterization of Clostridium butyricum neurotoxin and its trypsin-induced fragment by use of monoclonal antibodies against Clostridium botulinum type E neurotoxin.
We examined the reactivities of Clostridium butyricum neurotoxin to nine monoclonal antibodies against Clostridium botulinum type E neurotoxin which recognize the light chain or the amino-terminal half (H-1 fragment) or the carboxyl-terminal half (H-2 fragment) of the heavy chain of botulinum neurotoxin. Butyricum neurotoxin and its derived chains did not react to two of four monoclonal antibodies recognizing the light chain, one of three recognizing the H-1 fragment, and one of two recognizing the H-2 fragment. The results indicate that the immunological difference between the two neurotoxins is not attributable to a particular portion of the toxin molecule. The fragment of butyricum neurotoxin obtained by prolonged tryptic treatment was found to comprise the light chain and H-1 fragment linked together by a disulfide bond.
Animals↗
Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D.
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Base Sequence↗
ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells.
Botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) was purified to homogeneity and added to cultured rat pheochromocytoma PC-12 cells. Incubation with this exoenzyme caused inhibition of cell growth and induced neurites as well as acetylcholine esterase in these cells. These changes were dependent on the amount of the enzyme added to the culture, which correlated with the in situ ADP-ribosylation of the rho/rac proteins in the cells. Preincubation with a specific anti-C3 exoenzyme monoclonal antibody inhibited both the ADP-ribosyltransferase activity and the neurite-inducing activity of the enzyme preparation. These results suggest that C3 exoenzyme affected the cellular function of the rho/rac proteins by ADP-ribosylation to induce these changes in the cells.
Acetylcholinesterase↗
Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan.
Four strains of Clostridium botulinum type A having been associated with infant botulism in Japan, and another strain isolated from honey not associated with infant botulism, were found to be hemagglutinin (HA) negative. These strains do not produce L (Mr 500 kDa) nor LL toxin (Mr 900 kDa) but M toxin (Mr 300 kDa) only. No marked difference was found between the HA-positive and HA-negative strains in other biochemical properties, but the HA-negative strains tended to colonize more easily in the intestines of infant mice than did HA-positive strains. The toxin of HA-positive strains and that of HA-negative strains differed in the antigenicity of part of the toxic component and that of the nontoxic component, and in the molecular size of the toxic component.
Botulinum Toxins↗
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↗
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↗
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↗
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↗
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↗
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↗
Morphological effects of Clostridium botulinum C3 exoenzyme on cultured cells.
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ADP Ribose Transferases↗