Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D.
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Base Sequence↗
Biomedical subjects
S Kozaki
Publications and source records attributed to S Kozaki.
Hemagglutinating and binding properties of botulinum C2 toxin.
To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition. Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II. Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones. On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes. Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose. Thyroglobulin and bovine salivary mucin were much stronger inhibitors. From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).
Acetylgalactosamine↗
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↗
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↗
[Prevention and control of nosocomial infection caused by methicillin resistant Staphylococcus aureus in premature infant ward--prevention effect of "povidone iodine solution" wipe of neonatal skin].
In January 1983, a number of premature infants under management in the premature infants' unit of our hospital were found to have bacteremia due to Staphylococcus aureus. By the end of February of the same year, 4 of these infants, who had been treated in the same unit, developed impetigo. The S. aureus responsible for this condition was classified as type IV by a coagulase typing. In a subsequent antimicrobial susceptibility test using the disk diffusion method, this microorganism was found to be resistant to methicillin, erythromycin and lincomycin, and to be susceptible to tetracycline, chloramphenicol and cefmetazole, indicating that it was a methicillin resistant S. aureus (MRSA). Because the result from the coagulase typing agreed with the antimicrobial susceptibility pattern in all cases, we concluded that these cases represented nosocomial infection with MRSA. The source and route of the infection were investigated, and measures taken to prevent bacterial spread from carriers and to keep instruments and environments clean. Although the source of infection was not identified. Then, we tried wiping the body surface of the premature infants with an Isodine solution (10% PVP-I, 1:100 dilution) in order to prevent colonization of the microorganism on the body surface. With this application+, MRSA was no longer detected from the body surface of the premature infants, and no additional MRSA infection occurred in the premature infants' unit. Data collected for premature infants' managed at our hospital in the subsequent 6 years allows us to conclude that MRSA infection can be almost completely controlled by frequent surveys of carriers and appropriate body surface wiping with Isodine solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Cross Infection↗
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↗
Isolation and characterization of a novel human monoclonal antibody that neutralizes tetanus toxin.
A human monoclonal antibody, designated 53-2-4, has been isolated and characterized in terms of its ability to interact with clostridial neurotoxins. In enzyme-linked immunosorbent assay assays the antibody reacted with native tetanus toxin, tetanus toxoid and the C fragment obtained from the carboxyterminus of the toxin (AA 864-1314). The antibody did not react with the B fragment of tetanus toxin (AA 1-863) or with six serotypes of botulinum neurotoxin (A to F). Both culture supernatant from the clonal line producing the antibody as well as homogeneous protein obtained by affinity purification of the antibody neutralized tetanus toxin. When tested in vivo, the antibody provided complete production against a supralethal injection of toxin; when tested in vitro, the antibody produced at least 99% inactivation of a 1 x 10(-9) M solution of toxin. The exceptional neutralizing activity of the antibody was attributed to its high affinity for the toxin (4.2 x 10(-10) mol/liter). Animal experiments revealed a novel phenomenon that has been labeled delayed intoxication. At the appropriate ratio of antibody to antigen, the toxin was retained in the host in a latent form. After several days the biological activity of the toxin became apparent and there was onset of nervous system poisoning. Isolated tissue experiments showed that each antibody molecule is capable of associating with two antigen molecules. The antibody has greater neutralizing activity when mixed with free toxin than when mixed with toxin already bound to plasma membrane receptors.
Animals↗
[Molecular structure and function of Clostridium botulinum neurotoxin].
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Acetylcholine↗
ADP-ribosylation of the rho/rac gene products by botulinum ADP-ribosyltransferase: identity of the enzyme and effects on protein and cell functions.
1. Botulinum C1 toxin and C3 exoenzyme were purified from the culture filtrate of type C Clostridium botulinum strain 003-9, and specific antibodies were raised against each protein. Immunochemical analysis using these antibodies revealed the presence of minute amount of a C3-like molecule in C1 toxin preparation which tightly binds to the toxin component(s). This enzyme complex was separated from the major neurotoxin. Thus, the ADP-ribosyltransferases in C1 and D toxins and C3 exoenzyme appear to come from the same origin, and should be called together botulinum C3 enzyme. 2. Botulinum C3 enzyme ADP-ribosylates the rho and rac gene products, a family of small molecular weight GTP-binding proteins homologous to ras p21s. This ADP-ribosylation occurs at Asn41 of the rho products which is located in their putative effector domain, suggesting that it interferes interaction of these GTP binding proteins with their effector molecules. 3. When incubated with PC-12 cells, the enzyme inhibits cell growth and induces neurites and acetylcholine esterase. Several lines of evidence suggest that the ADP-ribosylation of the rho/rac proteins is responsible for these changes.
ADP Ribose Transferases↗
Morphological effects of Clostridium botulinum C3 exoenzyme on cultured cells.
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ADP Ribose Transferases↗
Use of monoclonal antibodies as probes for the structure and biological activity of botulinum neurotoxin.
Experiments were done to help clarify the structure-function relationships that govern the interaction between botulinum neurotoxin and the cholinergic neuromuscular junction. Work was done with type E toxin in three different states: 1) unactivated (post-translational product before proteolytic processing), 2) activated (proteolytically modified product) and 3) denatured. Four different monoclonal antibodies were studied (E3, E14, E17 and E32), three of which were capable of diminishing the potency of the toxin. All four antibodies had approximately equivalent affinity for the unactivated and the activated forms of the toxin. Monoclonals E17 and E32 had little ability to interact with denatured toxin, suggesting they recognized conformational epitopes; monoclonals E3 and E14 retained partial ability to bind to denatured toxin, suggesting they recognized both conformational and linear determinants. When phrenic nerve-hemidiaphragm preparations were exposed to toxin under conditions that allowed binding but retarded internalization, the toxin remained accessible to antibodies. However, when tissues were stimulated in an effort to promote endocytosis, the toxin disappeared from accessibility to antibodies. The data indicate that various antigenic domains remain exposed after binding and suggest that certain parts of the toxin molecule undergo little or no conformational change during binding. The data further indicate that the molecular domains recognized by E14, E17 and E32 are internalized simultaneously.
Animals↗
[Bone marrow suppression in gynecological patients with malignant tumor treated with combination chemotherapy cyclophosphamide, pirarubicin and cisplatin].
We treated 14 patients with cyclophosphamide, adriamycin and cisplatin (CAP), and 16 patients with cyclophosphamide, Pirarubicin and cisplatin (CTP). Hematological changes in the peripheral blood were compared in the two groups to determine whether there was any difference in bone marrow suppression. 1) The lowest leukocyte counts were similar in the two groups. The leukocyte count reached its nadir at 11.6 +/- 2.1 days in the CTP group and at 15.1 +/- 2.8 days in the CAP group, a significant difference (p less than 0.01). 2) The leukocyte count recovered rapidly in the CTP group and was significantly higher (p less than 0.01) at 3 to 4 weeks than in the CAP group, and it returned to the pretreatment level in the CTP group in the fourth week. 3) The platelet count reached its lowest level in the second week in both groups. In the CTP group, it was significantly higher (p less than 0.01) than in the CAP group. 4) Reticulocyte count reached its lowest level in the first week in both groups, and then started increasing. 5) In the CTP group, a course of treatment was 28.0 +/- 2.3 days and it was 29.5 +/- 2.3 days in the CAP group. In 28% of the CPA group, it took 30 days or more for the leukocyte count to return to normal after one course, while none of the CTP group did the leukocyte count remain low for 30 days. These results show that CTP causes more rapid bone marrow suppression (particularly leukocytopenia) than CAP, does but that a recovery is more rapid with CTP. These CTP may be a better form of treatment than CAP.
Antineoplastic Combined Chemotherapy Protocols↗
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↗
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↗
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↗
Production of monoclonal antibody to characterize the antigen of Paragonimus westermani.
We produced monoclonal antibodies against the adult antigen of Paragonimus westermani to investigate the expression of the stage-specific antigen of adult flukes. Two hybridoma cell lines, A1-2 and A4-1, were established. A1-2 reacted specifically only with the adult antigen in an enzyme-linked immunosorbent assay (ELISA), whereas A4-1 reacted with both adult and larval antigens. By immunoblotting analysis, A1-2 was found to react with four bands with molecular weights of 35,000, 17,000, 15,500, and 12,500 in the adult antigen but with none in the larval antigen. A4-1 was found to bind to the band of 27,000 daltons in the adult antigen as well as those of 28,000 and 26,000 daltons in the larval antigen. By immunohistochemical methods, a positive reaction was observed only in the parenchymal tissues of the adult flukes with A1-2. A4-1 reacted with the substance that located on the gut epithelium and in the luminal contents of both the adult and larval flukes. These results indicate that the adult P. westermani possesses an antigen or antigenic determinant specific to the adult stage, as well as that common to both the larval and adult stages.
Animals↗
Ultrasound and CT diagnosis of intramural duodenal hematoma.
An 8-year-old boy with traumatic duodenal hematoma is reported. Diagnosis is easy if one is familiar with its typical findings in modern imaging modalities.
Child↗