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H Kurazono

Publications and source records attributed to H Kurazono.

69 records · Page 4Linked to original sources
PubMed1993

Identification and characterization of heat-stable enterotoxin II-producing Escherichia coli from patients with diarrhea.

Stock strains of Escherichia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-producing enterotoxigenic E. coli can cause human diarrhea.

Animals↗
PubMed1993

Cloning and sequencing of two new Verotoxin 2 variant genes of Escherichia coli isolated from cases of human and bovine diarrhea.

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.

Animals↗
PubMed1992

Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.

To define conserved domains within the light (L) chains of clostridial neurotoxins, we determined the sequence of botulinum neurotoxin type B (BoNT/B) and aligned it with those of tetanus toxin (TeTx) and BoNT/A, BoNT/C1, BoNT/D, and BoNT/E. The L chains of BoNT/B and TeTx share 51.6% identical amino acid residues whereas the degree of identity to other clostridial neurotoxins does not exceed 36.5%. Each of the L chains contains a conserved motif, HExxHxxH, characteristic for metalloproteases. We then generated specific 5'- and 3'-deletion mutants of the L chain genes of TeTx and BoNT/A and tested the biological properties of the gene products by microinjection of the corresponding mRNAs into identified presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Toxicity was determined by measurement of neurotransmitter release, as detected by depression of postsynaptic responses to presynaptic stimuli (Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844-7848). Our studies allow the following conclusions. 1) Residues Cys439 of TeTx and Cys430 of BoNT/A, both of which participate in the interchain disulfide bond, play no role in the toxification reaction. 2) Derivatives of TeTx that lacked either 8 amino- or 65 carboxyl-terminal residues are still toxic, whereas those lacking 10 amino- or 68 carboxyl-terminal residues are nontoxic. 3) For BoNT/A, toxicity could be demonstrated only in the presence of added nontoxic heavy (H) chain. A deletion of 8 amino-terminal or 32 carboxyl-terminal residues from the L chain had no effect on toxicity, whereas a removal of 10 amino-terminal or 57 carboxyl-terminal amino acids abolished toxicity. 4) The synergistic effect mediated by the H chain is linked to the carboxyl-terminal portion of the H chain, as demonstrated by injection of HC-specific mRNA into neurons containing the L chain. This finding suggests that the HC domain of the H chain becomes exposed to the cytosol during or after the putative translocation step of the L chain.

Acetylcholine↗
PubMed1991

Clostridial neurotoxins: from toxins to therapeutic tools?

Tetanus toxin and botulinum toxins are powerful neurotoxins which block neurotransmitter release through an unknown mechanism my means of their light chains. The heavy chains provide the machinery for neuroselective binding, internalization, retrograde intraaxonal transport, and translocation of the L-chains into the cytosole. We have cloned and sequenced the structural genes of tetanus toxin and of five serologically distinct botulinum toxins to identify structurally and functionally conserved subdomains. The minimum essential domains of the L-chains of tetanus and botulinum toxin type A were identified by combined in vitro transcription and microinjection of L-chain specific mRNA into identified presynaptic neurons of Aplysia californica. In addition, a nontoxic mutant of tetanus was generated by replacing histidine(237) by a proline residue. The development of nontoxic neuroselective transporter molecules carrying various marker enzymes is discussed.

Amino Acid Sequence↗
PubMed1990

The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins.

The seven serologically different botulinum neurotoxins are highly potent protein toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The activated toxins consist of the toxifying A-subunits (Mr approximately 50,000) linked by a disulfide bond to the receptor-binding BC-subunits (Mr approximately 100,000). We have established the complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr = 149,425) and a partial sequence of botulinum neurotoxin type E (273 amino acid residues) as deduced from the corresponding nucleotide sequences of the chromosomally located structural genes. The promoter of the BoNT/A gene is inactive in Escherichia coli. Primer extension experiments indicated that initiation of transcription of the BoNT/A gene occurred 118 nucleotides upstream from the ATG codon. A comparison of the protein sequence revealed an overall identity of 33.8% to that of tetanus toxin. No significant similarity to other known proteins including ADP-ribosylating toxins could be detected. Three of the six histidine residues of the A-subunit of BoNT/A were found in the peptide sequence H223ELIHXXH230 within a domain of predicted alpha-helical secondary structure. This motif is also found in similar positions of the A-subunits of tetanus toxin and BoNT/E.

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome.

Cellular DNA extracted from Escherichia coli strain B2F1 (O91:H21) was found to contain two separate DNA sequences that hybridized with a Vero toxin 2 (VT2)-specific gene probe under stringent conditions. These two sequences were cloned and both were shown to encode a variant of Vero toxin 2 (VT2vh). The nucleotide sequences of the operons encoding VT2vh, designated as vtx2ha and vtx2hb, were determined. The two operons were nearly identical (99% overall DNA homology) and both encoded A subunits of 319 amino acid residues and B subunits of 89 amino acid residues, the A and B subunit genes being separated by a stretch of 14 bp. The A and B subunit genes of the vtx2ha operon exhibited 98.6% and 95.5% DNA homology, respectively, with those of the slt-II operon encoding Shiga-like toxin II (or VT2) cloned from a strain from a patient with hemorrhagic colitis, while the A and B subunit genes of the vtx2ha operon showed 94.5% and 82.8% DNA homology, respectively, with those of the slt-IIv operon encoding a SLT-II variant cloned from a strain isolated from a pig with edema disease. The nucleotide sequences of the presumed promoters and presumptive ribosome binding sites in the vtx2ha, vtx2hb, and slt-II, and slt-IIv operons were identical. These results indicate that nucleotide sequences encoding a family of VT2-related toxins are present in various strains of E. coli and that the sequences of the genes for A subunits are better conserved than those of the B subunit genes.

Amino Acid Sequence↗
PubMed1990

Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons.

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons. If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context. To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations. Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known. Both toxins block neurotransmitter release at nanomolar intracellular concentrations. These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced. Their active sites are located (or partly located) on the light chain. mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ.

Animals↗
PubMed1990

Molecular biology of Clostridial toxins: expression of mRNAs encoding tetanus and botulinum neurotoxins in Aplysia neurons.

mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of Aplysia californica. The size of the current response measured in the voltage clamped postsynaptic neuron was taken as indicator of the quantity of acetylcholine released. Depression of neurotransmitter release similar to that observed when native light chains of the two toxins were injected but needing an additional delay of 30 to 40 minutes, demonstrated a successful expression of a foreign mRNA injected into a neuron in situ.

Acetylcholine↗
PubMed1989

Expression of tetanus toxin subfragments in vitro and characterization of epitopes.

To define epitopes of tetanus toxin, we compared four different in vitro systems in terms of their ability to produce tetanus toxin-specific subfragments from cloned DNA. A transcription-translation system developed from a nontoxigenic strain of Clostridium tetani was found to yield predominantly full-sized peptides. Such peptides were used to map six different epitopes for eight monoclonal antibodies. The toxin-neutralizing properties of the antibodies were determined in an in vitro assay, based on the toxin-mediated inhibition of norepinephrine release from rat brain particles. Two monoclonal antibodies recognizing epitopes within the regions Ser-744 to Ser-864 and Ile-1224 to Asp-1315 could neutralize the toxin. A third nonneutralizing antibody was shown to recognize the synthetic peptide Phe-947 to Glu-967 derived from the tetanus toxin sequence. This peptide contains a human T-cell epitope.

Animals↗
PubMed1988

Identity of molecular structure of Shiga-like toxin I (VT1) from Escherichia coli O157:H7 with that of Shiga toxin.

The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests. The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures. The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage. This nucleotide sequence was different from that reported by Jackson et al. (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al. (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al. (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported. The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine. Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond.

Amino Acid Sequence↗
PubMed1987

Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken.

Fluid accumulation was induced in ligated intestinal loops of both pheasants and chickens injected with crude Clostridium botulinum C2 toxin. Necrosis of the epithelium and lamina propria of the duodenum and jejunum occurred in both species of bird after intraduodenal administration of crude C2 toxin. The severity of such reactions depended upon the dose and the period after administration of C2 toxin up to eight hours, and such reactions were suppressed specifically with rabbit anti-C2 toxin. These results confirm that C2 toxin is one of the causes of diarrhoea in avian botulism.

Animals↗
PubMed1985

Botulism among penned pheasants and protection by vaccination with C1 toxoid.

Fifty to 100 per cent of about 8000 penned pheasants raised on a farm in Hiroshima Prefecture died annually for three years. Deaths were ascribed to type C botulism. Vaccination of four groups of pheasants with partially purified C1 toxoid effectively protected them against type C botulism. The protective efficacy of the toxoid was emphasised by the relatively high susceptibility of the pheasant to C1 toxin.

Animals↗