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Biomedical subjects

Y Kamata

Publications and source records attributed to Y Kamata.

At least 109 records · Page 6Linked to original sources
PubMed1990

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↗
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↗
PubMed1988

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↗
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↗
PubMed1987

Type 1a glycogen storage disease with hepatoblastoma in siblings.

Clinical and histologic details of the two siblings with type 1a glycogen storage disease (GSD-1a) who developed hepatoblastoma are presented. The light microscopic studies on hepatic tumor in both siblings revealed fetal type of hepatoblastoma. Ultrastructural findings in Patient 2 showed markedly altered mitochondria, which were frequently surrounded by the rough endoplasmic reticulum. This is the first known occurrence with this association, and the third report on the familial occurrence of this neoplasm. Glycogen storage disease may increase the risk of hepatocellular carcinoma and hepatoblastoma.

Adult↗
PubMed1987

Antigenic structure of Clostridium botulinum type B neurotoxin and its interaction with gangliosides, cerebroside, and free fatty acids.

A fragment distinct from the heavy and light chains was obtained by treatment of Clostridium botulinum type B neurotoxin with chymotrypsin. Enzyme-linked immunosorbent assay and immunoblotting analysis with monoclonal antibodies showed that the fragment consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond. Monoclonal antibodies reacting to the heavy chain but not to the fragment were thought to recognize the epitopes on the remaining portion (H-2 fragment) of the heavy chain, being easily digested by chymotrypsin. Thus, the antigenic structure of type B neurotoxin resembles those of type A and E neurotoxins. The chymotrypsin-induced fragment bound to cerebroside and free fatty acids but not to gangliosides. The manner of binding of type B neurotoxin to gangliosides and free fatty acids was different from those of type A and E neurotoxins. Such differences in the reactivities to lipids may be related to the finding that each neurotoxin binds to a type-specific site on the neural membrane.

Antibodies, Monoclonal↗
PubMed1987

Activities of Aeromonas hydrophila hemolysins and their interaction with erythrocyte membranes.

The activity of hemolysin produced by Aeromonas hydrophila CA-11, isolated from an environmental source, was more sensitive to temperature than that of hemolysin produced by strain AH-1, isolated from a diarrheal case. CA-11 hemolysin failed to elicit hemolysis below 10 degrees C. Immunoblotting analyses showed that both hemolysins formed into oligomers in rabbit erythrocyte membrane even when no hemolysis occurred. suggesting that the binding and the subsequent oligomerization are temperature independent. Sodium salicylate inhibited lysis of rabbit erythrocytes by both hemolysins, but selected monosaccharides and oligosaccharides did not. Thin-layer immunostaining indicated that both hemolysins bound to phosphoglycerides with net negative charge but weakly to the ones with no net negative charge. Neither sphingomyelin nor lysophosphoglyceride reacted with the hemolysins, whereas the hemolysins bound to free fatty acids. These results suggest that the binding of either hemolysin to the membrane component, probably phospholipid, requires both negative charge of the polar head group and suitable hydrophobicity of the nonpolar tails.

Aeromonas↗
PubMed1986

Evidence for direct binding of Clostridium botulinum type E derivative toxin and its fragments to gangliosides and free fatty acids.

Clostridium botulinum type E derivative toxin and its heavy chain bound to gangliosides GT1b, GD1a and GQ1b and saturated and unsaturated free fatty acids with chain lengths of 14-20 carbons. The L-H-1 fragment lacking the carboxyl-terminal portion of the heavy chain bound to free fatty acids but not to gangliosides. These observations led us to a new hypothesis on the mechanism of binding between botulinum toxin and gangliosides; the carboxyl-terminal portion (H-2 fragment) of the heavy chain binds to an oligosaccharide residue of gangliosides and then the amino-terminal portion (H-1 fragment) interacts with the hydrophobic portion of gangliosides consisting of fatty acids.

Binding Sites↗
PubMed1986

Production and characterization of monoclonal antibodies to Clostridium perfringens enterotoxin.

Four hybridoma cell lines producing monoclonal antibodies to Clostridium perfringens enterotoxin were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with the enterotoxin and its toxoid. An enzyme-linked immunosorbent assay indicated that the two antibodies, 2-B-4 and 3-G-10, bound to those regions that were located close each other; the others, 3-B-2 and 2-H-2, bound to other independent regions on the enterotoxin. Release of 51Cr from Vero cells with the enterotoxin was inhibited by either 2-B-4 or 3-G-10, both of which inhibited the binding of 125I-labeled enterotoxin to the cells. Neither binding nor cytotoxicity of the enterotoxin was affected by 2-H-2; 3-B-2 only barely inhibited the binding but neutralized the enterotoxin shown by 51Cr release. It seems justified to conclude that 3-B-2 blocks the toxic action after the enterotoxin has bound to Vero cells.

Animals↗
PubMed1986

The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin.

Six monoclonal antibodies against Clostridium botulinum type E derivative toxin were prepared. Three of the five binding to the heavy chain neutralized the derivative toxin; the other one binding to the light chain did not. Immunoblotting analysis with the monoclonal antibodies showed that the fragment obtained by tryptic digestion consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond(s) and that the antigenic determinants common between type E and F derivative toxins were located on both the heavy and light chains. The fragment induced by chymotrypsin treatment, like the tryptic fragment, bound to four monoclonal antibodies. The mild tryptic treatment and reduction resulted in separation of the chymotryptic fragment into two smaller fragments corresponding to the light chain and H-1 fragment. These results indicate that H-1 fragment contains the amino-terminal portion of the heavy chain. The monoclonal antibody neutralizing the toxin and probably recognizing the epitope on the carboxyl-terminal portion (H-2 fragment) of the heavy chain effectively competed for binding of 125I-labeled derivative toxin to synaptosomes. Of the two monoclonal antibodies neutralizing the toxin and recognizing the epitopes on H-1 fragment, one partially inhibited binding, but the other did not. This suggests that the binding of 125I-labeled derivative toxin depends mainly on the carboxyl-terminal region of the heavy chain and that interference with binding is not the only means of toxin neutralization.

Animals↗
PubMed1985

Inhibitory effect of estradiol-17 beta and progesterone on bactericidal activity in uteri of rabbits infected with Escherichia coli.

The influence of ovarian hormones at different estrous stages on the bactericidal activity of the uterus in rabbits was investigated. When Escherichia coli cells were inoculated in ligated uteri, the survival period of the bacteria in the uterus at the luteal phase was clearly longer than that at the follicular phase. At the luteal phase, high levels of plasma estradiol-17 beta and progesterone were detected. A luteolytic treatment with prostaglandin F2 alpha and human chorionic gonadotropin at the luteal phase lowered plasma progesterone levels and prompted bacterial clearance from the uterus. In ovariectomized rabbits, E. coli from the uterine exudates was not detected 6 days after the inoculation in both the nontreated and estradiol-17 beta-treated animals. In the progesterone-treated rabbits, the survival period of E. coli was longer than that in the nontreated and estradiol-17 beta-treated animals. When estradiol-17 beta and progesterone at the ratio of 1:100 were administered concurrently, E. coli survived for the longest period in the rabbits treated with various doses of different hormones. Formalin-killed E. coli cells were inoculated into the uterine lumen, and 4 h later the proportion of heterophils phagocytizing the bacteria dropped in the progesterone-treated rabbits and in the estradiol-17 beta- and progesterone-treated rabbits, but there was no significant difference in heterophil numbers among the rabbits treated with different hormones. The present results suggest that progesterone inhibits the bactericidal activity of the uterus and that estrogen concurrently secreted at the luteal phase promotes the inhibitory action of progesterone, although estrogen alone hardly affects the uterine defense. In addition, the lowering of the bactericidal activity of the uterus at the luteal phase may be attributable to lower activity of phagocytosis by heterophils infiltrated into the uterine lumen.

Animals↗