Biomedical subjects

T Miwatani

Publications and source records attributed to T Miwatani.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1.

Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.

Amino Acid Sequence↗

PubMed1992

Comparison of Vibrio parahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates.

TDH-related hemolysin (Vp-TRH) produced by Kanagawa-phenomenon-negative (KP-) Vibrio parahaemolyticus has been demonstrated to be a possible virulence determinant. Though almost half of KP- isolates examined from diarrhoeal patients produced Vp-TRH, few reports mentioned the ability of environmental isolates to produce Vp-TRH. Considering the route of infection with V. parahaemolyticus, this toxin must be produced by the organisms in the sea or in sea food. To confirm that Vp-TRH produced by V. parahaemolyticus could be involved in sea-food-borne diarrhoeas, Vp-TRH-producing strains were isolated from the environment, identified and hemolysin purified from these strains was compared to hemolysin (Vp-TRH) isolated from diarrhoeal patients. The results showed that the hemolytic activity, antigenicity, reactivity in the rabbit ileal loop test and N-terminal amino acid sequence of Vp-TRH from environmental strains was indistinguishable from the toxin of clinical origin.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

A survey of urease-positive Vibrio parahaemolyticus strains isolated from traveller's diarrhea, sea water and imported frozen sea foods.

The frequency of urease-positive Vibrio parahaemolyticus among isolates from patients, imported frozen sea foods and the environment (sea water) was studied. The highest isolation frequency of urease-positive V. parahaemolyticus was found in clinical isolates (11.2% out of 204 strains examined). Urease-positive V. parahaemolyticus was found in 5.7% of 88 frozen sea food-isolates examined, but no strains isolated from sea water were urease-positive. The isolates were further examined for the production of thermostable direct hemolysin (Vp-TDH) and its related hemolysin (Vp-TRH). Both are possible pathogenic toxins produced by mostly clinical isolates of V. parahaemolyticus. Urease-positive strains have a tendency to associate with clinical isolates producing both or neither Vp-TDH and Vp-TRH. Rabbit ligated ileal loops test was performed with several strains of urease-positive and -negative clinical isolates, and we found that some strains producing urease, even those which do not produce Vp-TDH or Vp-TRH, caused intestinal fluid accumulation.

Diarrhea↗

PubMed1992

A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody.

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.

Antibodies, Monoclonal↗

PubMed1992

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.

Alkaline Phosphatase↗

PubMed1992

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.

Erythrocytes↗

PubMed1991

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus.

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.

Bacterial Proteins↗

PubMed1991

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus.

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.

Bacillus cereus↗

PubMed1991

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus.

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.

Amino Acid Sequence↗

PubMed1991

Production of monoclonal antibodies against a hemagglutinin/protease of Vibrio cholerae non-01.

Two hybridoma cell lines producing monoclonal antibodies (MAbs) against a hemagglutinin/protease (HA/P) from Vibrio cholerae non-01 were produced and characterized. The two MAbs contained the kappa light chain and were IgG1 type. They similarly neutralized HA/P protease activity derived from both V. cholerae non-01 and V. cholerae 01, whereas they were unable to neutralize the hemagglutinating activity of HA/P, suggesting that the epitopes for protease and hemagglutination activities are different. Western blotting analysis and the cross-neutralization test with the two MAbs confirmed the identity of HA/P produced by V. cholerae non-01 and 01. This study also suggests that HA/P of V. cholerae and a protease of V. parahaemolyticus are immunologically unrelated.

Animals↗

PubMed1991

Detection and purification of the free A subunit of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.

After removal of total B subunit and heat-labile enterotoxin (LT) from crude cell extracts of enterotoxigenic Escherichia coli (HB 101-EWD 299) by Bio-gel A 5 m column chromatography, the crude cell extract was shown to contain a free A subunit (A' subunit) that did not bind to the coligenoid of the B subunits. The A' subunit was found to be immunologically identical to the A subunit of holo-LT and was purified to show only one band in SDS-poly-acrylamide gel electrophoresis (PAGE). The mobility of the A' subunit was identical to that of the A subunit of holo-LT. The pI value of the A' subunit was also the same as that of the A subunit of holo-LT. These data suggest that in enterotoxigenic E. coli there is free A subunit which may be involved in formation of holo-LT, analogously to free B subunit (coligenoid), and that the free A subunit is physicochemically and immunologically identical to the A subunit of holo-LT.

Bacterial Toxins↗

PubMed1991

Detection of a heat-labile enterotoxin gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes.

Two alkaline phosphatase-conjugated 24-mer oligonucleotide probes were developed to detect the heat-labile enterotoxin gene in enterotoxigenic Escherichia coli. Probes were antisense codon sequences, which are transcribed into mRNA, of the heat-labile enterotoxin gene of enterotoxigenic Escherichia coli of human origin. Using dot-blot hybridization, probes were tested with 100 clinical isolates and evaluated by a reflectance-type densitometer. Results agreed very well with those of an immunological test, the Biken test, and a 32P-labelled recombinant DNA probe. The oligonucleotide probes did not react with nucleic acids prepared from other diarrhoeagenic bacterial pathogens. Thus, the alkaline phosphatase-conjugated oligonucleotide probes seem to be highly sensitive and specific for detection of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli. Moreover, the results indicate a potential usefulness for densitometric evaluation of DNA hybridization.

Alkaline Phosphatase↗

PubMed1991

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus.

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.

Acetic Anhydrides↗

PubMed1991

Characterization of thermostable direct hemolysins encoded by four representative tdh genes of Vibrio parahaemolyticus.

Four tdh genes encoding thermostable direct hemolysin (TDH) cloned from two representative strains of V. parahaemolyticus (tdh 1 and tdh 2 from a hemolytic strain, tdh3 and tdh4 from a non-hemolytic strain) have different nucleotide sequences (the maximum divergence: 3.3%). In this study, each tdh gene product was purified from the lysate of Escherichia coli cells carrying the cloned gene and their properties were compared to investigate the influence of the amino acid substitutions caused by these base changes. The four tdh gene products showed different electrophoretic mobilities under non-denaturing conditions. All the gene products had hemolytic activities for various animal erythrocytes, stimulated vascular permeability in the rabbit skin, and were lethal to mice, although their potencies were slightly different. Antigenicities of the four gene products were indistinguishable. These results indicate that the four tdh genes have evolved to maintain a fundamental molecular structure and biological activities of the gene products and minor structural and/or charge differences of the molecules are perhaps responsible for the slight divergence of their biological activities.

Animals↗

PubMed1991

Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus.

The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.

Amino Acid Sequence↗

PubMed1991

[Bacteriological examination of infections in the field of oral surgery].

Etiology of bacterial infections in the field of oral surgery was studied. A total of 270 samples collected from patients with encapsulated abscess in their oral cavities was examined and bacteria were isolated from the 244 samples (90.4%). The following results were found; 1) Organisms more than one from one sample were frequently isolated from cases with parodontitis, pericoronitis and gnathitis. Isolation of anaerobic bacteria was common (54.2%). 2) Streptococcus milleri and Streptococcus sanguis and Capnocytophaga species were the most common isolates among aerobic gram-positive and gram-negative bacteria, respectively. 3) Peptostreptococcus micros and Eubacterium lentum were most frequent isolates among gram-negative anaerobic bacteria. Among gram-negative bacteria, Oral Group Bacteroides, especially Bacteroides gingivalis, Bacteroides intermedius, Bacteroides buccae and Bacteroides oralis were most prominent. 4) Isolation frequency of bacteria (both species and strains) was high from samples obtained from patients before antibiotic chemotherapy. 5) Most strains were sensitive to Midecamycin acetate and Josamycin. Minimum inhibitory concentration of 80% isolates (MIC80) against these antibiotics was 0.39 microgram/ml.

Bacteria↗

PubMed1991

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Purification and characterization of a new heat-stable enterotoxin produced by Vibrio cholerae non-O1 serogroup Hakata.

The possible production of a heat-stable enterotoxin (Vc-H-ST) by Vibrio cholerae non-O1 serogroup Hakata was investigated, and the purified Vc-H-ST was characterized. It has a unique amino acid sequence, LIDCCEICCNPACFGCLN. This sequence is quite similar to that of the heat-stable enterotoxin (NAG-ST) produced by V. cholerae non-O1 except for one amino acid (leucine) residue excess at the N terminus. Other characteristics, including biological activity, are compatible with those of NAG-ST.

Amino Acid Sequence↗