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H Kurazono

Publications and source records attributed to H Kurazono.

At least 55 records · Page 3Linked to original sources
PubMed1995

Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction.

Primers to amplify the genes encoding the virulence factors of uropathogenic Escherichia coli, such as pilus associated with pyelonephritis (pap), haemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnf1) genes, were designed. The above primers along with previously reported primers for S fimbriae (sfa) and afimbrial adhesin I (afaI) genes were combined to develop a multiplex polymerase chain reaction (PCR) for detection of the respective virulence factors and for the identification of uropathogenic E. coli. The multiplex PCR to detect pap, sfa, afaI, hly, aer and cnf1 genes was highly specific and the sensitivity was found to be about 5 x 10(3) colony forming units of E. coli per ml. A total of 194 E. coli strains isolated from patients with simple acute cystitis were examined by the multiplex PCR and the results were in complete agreement with that obtained by DNA colony hybridization test. The multiplex PCR developed was, therefore, concluded to be a useful, sensitive and rapid assay system to identify uropathogenic E. coli.

Base Sequence↗
PubMed1995

Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal.

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.

Bacteriological Techniques↗
PubMed1995

[Detection of bacterial protein toxins by a bead-ELISA].

A highly sensitive bead-enzyme-linked immunosorbent assay to detect bacterial protein toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as a substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The sensitivities of the bead-ELISA for various bacterial protein toxins were as follows: less than 40 pg/ml for cholera enterotoxin (CT), less than 20 pg/ml for VT1 and less than 6 pg/ml for VT2 of enterohemorrhagic Escherichia coli. The bead ELISA was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. These data indicate that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples.

Acute Disease↗
PubMed1994

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin).

The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fimA, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.

Amino Acid Sequence↗
PubMed1994

Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content.

Amino Acid Sequence↗
PubMed1994

Specific detection of a verotoxin 2 variant, VT2vp1, by a bead-enzyme-linked immunosorbent assay.

A bead-enzyme-linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vp1, was developed. The sensitivity of the bead-ELISA was 200 pg/ml of the purified VT2vp1 and it did not react with 20 ng/ml of the purified VT2. The specificity of the bead-ELISA was examined with 107 strains of Verocytotoxin-producing Escherichia coli that include VT1-, VT2-, VT2vha-, VT2vhb- and VT2vp1-producing E. coli, and only VT2vp1-producing E. coli that were confirmed by VT2vp1-specific polymerase chain reaction gave positive results. It was noted that all 58 VT2vp1-producing E. coli strains were from pigs, but not from cows and humans.

Animals↗
PubMed1994

Construction of mutant genes for a non-toxic verotoxin 2 variant (VT2vp1) of Escherichia coli and characterization of purified mutant toxins.

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.

Animals↗
PubMed1994

Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal.

Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup. Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups. Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V. cholerae. Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins. All of the 223 representative strains of V. cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene. The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains. The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor. The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.

Animals↗
PubMed1993

Distribution of the zot (zonula occludens toxin) gene among strains of Vibrio cholerae 01 and non-01.

The distribution of the zot gene that encodes the zonula occludens toxin, a newly described toxin of Vibrio cholerae, among clinical, environmental and food isolates of V. cholerae 01 and non-01 was investigated. Both the zot gene and the ctx gene that encode cholera toxin were found in 247 of 257 clinical strains and 62 of 415 environmental or food isolates of V. cholerae 01. The zot gene, but not the ctx gene was found in 37 strains (one clinical strain and 36 environmental or food isolates). In addition, two of 31 clinical strains and six of 98 environmental or food isolates of V. cholerae' non-01 possessed both the zot gene and the ctx gene. These results demonstrated the predominantly concurrent occurrence of the zot gene and ctx genes among strains of V. cholerae 01 which suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea produced by V. cholerae 01.

Cholera Toxin↗
PubMed1993

Characterization of non-toxic mutant toxins of Vero toxin 1 that were constructed by replacing amino acids in the A subunit.

Three mutant toxins of Vero toxin 1 (VT1) produced by Escherichia coli strains carrying mutant VT1 genes that were constructed by oligonucleotide-directed site-specific mutagenesis were purified to homogeneity. They are E167Q (glutamic acid at position 167 from the N-terminus of the A subunit was replaced by glutamine), R170L (arginine at position 170 from the N-terminus of the A subunit was replaced by leucine) and E167Q-R170L (glutamic acid at position 167 and arginine at position 170 were replaced by glutamine and leucine, respectively). The purified E167Q and E167Q-R170L had markedly decreased activities, such as inhibition of protein synthesis, cytotoxicity to Vero cells and mouse lethality. The decrease in the R170L activities was less than those of the other two mutant VT1s. Neither additive nor synergistic decreases of the activities were observed in the double mutant E167Q-R170L in which two amino acids were replaced. Ouchterlony double gel diffusion showed that antiserum against the purified E167Q-R170L gave a line of identity between mutant VT1s and VT1. Moreover, anti-E167Q-R170L antiserum showed similar activity to that of anti-VT1 antiserum in neutralizing the Vero cell cytotoxicity and mouse lethality of VT1. The data suggest that these mutant VT1s, especially E167Q and E167Q-R170L, may be candidate toxoids that protect against diseases caused by Verocytotoxin-producing E. coli.

Animals↗
PubMed1993

Purification and some properties of a Vero toxin 2 (Shiga-like toxin II) variant (SLT-IIva) of Escherichia coli isolated from infantile diarrhea.

A variant of Vero toxin 2 (VT2), or Shiga-like toxin II (SLT-II) was purified from a cloned strain of Escherichia coli carrying a gene that encodes SLT-IIva, which is most closely related to SLT-IIv (VT2vp, VTe) in its nucleotide sequence. The purified SLT-IIva showed a slight difference from the purified SLT-IIv (VT2vp) in mobility on polyacrylamide gel disc electrophoresis and SDS-polyacrylamide gel slab electrophoresis and in pI, but both showed a similar potency in biological activities. Ouchterlony double diffusion test revealed that the purified SLT-IIva and SLT-IIv (VT2vp) formed a spur against anti-SLT-IIva and anti-SLT-IIv (VT2vp) antisera.

Animals↗
PubMed1993

Virulence patterns of Vibrio cholerae non-O1 strains isolated from hospitalised patients with acute diarrhoea in Calcutta, India.

A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989-1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32.1%), O11 and O34 (14.3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4-512. Fewer strains produced a cytotonic effect, with end-point titres of 2-16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14.3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35.7%). Ten (35.7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Detection of various variant verotoxin genes in Escherichia coli by polymerase chain reaction.

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.

Bacterial Toxins↗