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Biomedical subjects

M P Jackson

Publications and source records attributed to M P Jackson.

At least 37 records · Page 2Linked to original sources
PubMed1990

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli. Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon. Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene. The slt-IIv promoter was demonstrated to be identical to the slt-II promoter. The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters. The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter. In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E. coli strains with wild-type or mutant ferric uptake regulation (fur) alleles. Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene. A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB. These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron. Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.

Animals↗
PubMed1990

Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.

The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.

Amino Acid Sequence↗
PubMed1989

In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity.

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.

Bacterial Toxins↗
PubMed1989

Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.

Synthetic oligonucleotides, constructed from the nucleotide sequences of genes coding for the A subunit of Shiga-like toxin (SLT) I and the B subunit of SLT-II, were used as probes at different degrees of stringency to identify Escherichia coli producing different types of SLTs. At 45 degrees C, the A-I oligonucleotide probe hybridized with E. coli producing SLT-I, SLT-II, and variant of SLT-II (SLT-IIv). At 53 degrees C, only SLT-I-producing E. coli hybridized with this probe. At 45 degrees C, the B-II oligonucleotide probe hybridized with SLT-II- and SLT-IIv-producing E. coli. At 53 degrees C, this probe hybridized with only SLT-II-producing E. coli. The A-I and B-II oligonucleotide probes were subsequently tested for hybridization with 73 SLT-producing E. coli and 49 non-SLT-producing E. coli isolated in Asia and Canada. At 45 degrees C, the A-I oligomer had a sensitivity of 97% and a specificity of 100% in identifying SLT-producing E. coli. At 53 degrees C, the A-I oligonucleotide probe had a sensitivity of 92% and a specificity of 91% in identifying E. coli containing genes encoding SLT-I. At 45 degrees C, the B-II oligonucleotide had a 100% sensitivity and 97% specificity in identifying E. coli that hybridized with the SLT-II probe. Of 17 E. coli that hybridized only with the SLT-II probe, 10 did not hybridize with the B-II oligonucleotide at 53 degrees C. All 10 isolates were cytotoxic to Vero cells but not to HeLa cells, confirming that the B-II oligonucleotide probe used at 53 degrees C will differentiate isolates producing SLT-II and SLT-IIv.

Adult↗
PubMed1988

A high flow turbine CPAP system.

A continuous high flow CPAP system incorporating a turbine blower is described. The system achieves inspiratory flow rates of 150 l/min or more by means of reticulated gas flow and inspired oxygen fractions of 0.21-0.95. Positive airway pressure is provided by weighted disc valves and a modified aviation-type CPAP face mask provides electronic communication with the patient. The mobility of the system also enables its use as an intermittent physiotherapy aid. Work of breathing of the system, as assessed by total pressure fluctuations is at a minimum.

Acute Disease↗
PubMed1988

Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1.

The structural genes for Shiga toxin, designated stx A and stx B, were cloned from Shigella dysenteriae type 1 3818T, and a nucleotide sequence analysis was performed. Both stx A and stx B were present on a single transcriptional unit, with stx A preceding stx B. The molecular weight calculated for the processed A subunit was 32,225, while the molecular weight of the processed B subunit was 7,691. Comparison of the nucleotide sequences for Shiga toxin and Shiga-like toxin I (SLT-I) from Escherichia coli revealed that the genes for Shiga toxin and SLT-I were greater than 99% homologous; three nucleotide changes were detected in three separate codons of the A subunits. Only one of these codon differences resulted in a change in the amino acid sequence: a threonine in Shiga toxin at position 45 of the A subunit compared with a serine in the corresponding position in SLT-I. Furthermore, Shiga toxin and SLT-I had identical signal peptides for the A and B subunits, as well as identical ribosome-binding sites, a putative promoter, and iron-regulated operator sequences. These findings indicate that Shiga and SLT-I are essentially the same toxin. Southern hybridization studies with total cellular DNA from several Shigella strains and internal toxin probes for SLT-I and its antigenic variant SLT-II showed that a single fragment in S. dysenteriae type 1 hybridized strongly with the internal SLT-I probe. Fragments with weaker homology to the SLT-I probe were detected in S. flexneri type 2a but no other shigellae. No homology between the Shiga-like toxin II (SLT-II) probe and any of the Shigella DNAs was detected. Whereas SLT-I and SLT-II are phage encoded, no phage could be induced from S. dysenteriae type 1 or other Shigella spp. tested. These results suggest that the Shiga (SLT-I) toxin genes responsible for high toxin production are present in a single copy in S. dysenteriae type 1 but not in other shigellae. The findings further suggest that SLT-II genes are absent in shigellae, as are toxin-converting phages.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli.

A nucleotide sequence analysis was performed on the Shiga-like toxin I genes previously cloned from the Escherichia coli bacteriophage 933J. Two structural genes designated slt-I A and slt-I B were found to be oriented on a single transcriptional unit with slt-I A preceding slt-I B. A 12 base pair gap separated the in-phase open reading frames of slt-I A and slt-I B. Putative ribosome binding sites were identified 5' to both the slt-I A and slt-I B genes. Translation of the SLT-I nucleotide sequence revealed that both the A and B subunits were synthesized with signal peptides. The molecular weight calculated for the mature A subunit was 32,211 while the molecular weight of the mature B subunit was 7690.

Amino Acid Sequence↗
PubMed1986

Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus.

Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001.

Bacterial Toxins↗
PubMed1986

Sequence of the exfoliative toxin B gene of Staphylococcus aureus.

We sequenced the Staphylococcus aureus exfoliative toxin B gene contained on a 1.7-kilobase HindIII fragment of plasmid pRW001. The gene was located by comparison of the amino acid sequences of open reading frames with the amino-terminal sequence of exfoliative toxin B and the total amino acid composition of the protein (A.D. Johnson, L. Spero, J.S. Cades, and B.T. De Cicco, Infect. Immun. 24:679-684, 1979). The primary translation product consists of 274 amino acids and contains a 31-amino-acid N-terminal peptide presumably necessary for transport.

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of a bacteriophage factor that confers competence for genetic transformation to an exfoliative toxin-producing strain of Staphylococcus aureus.

Competence for genetic transformation in an exfoliative toxin-producing strain of Staphylococcus aureus was shown to be dependent on a virion factor that was isolated from a crude bacteriophage 80 alpha lysate. This competence-conferring factor was completely separated from infectious virus particles after either centrifugation through a neutral sucrose velocity gradient or fractionation on a Sepharose 2B gel. Since the competence-conferring factor tends to aggregate, optimal separation was obtained after treatment of the phage factor with the detergent Nonidet P-40. The competence-conferring factor had a molecular weight between 3 X 10(6) and 20 X 10(6) and an approximate sedimentation coefficient of 252. The factor was neutralized after interaction with antiserum prepared against isolated infectious 80 alpha virions. Electron microscopy of transforming cells that were exposed to isolated competence-conferring factor revealed a significant number of abnormally long and aggregated phage tail-like structures attached to the surface of recipient cells. This phenomenon was only observed in the presence of donor DNA, indicating that a phage tail-DNA-surface receptor complex might be one of the early steps in DNA-mediated transformation of S. aureus.

Bacterial Proteins↗
PubMed1979

Extraversion, neuroticism, and date of birth: a Southern hemisphere study.

The aim of this study was to determine whether variations in Eysenck Personality Inventory (EPI) scoring among zodiacal birth groups follow a similar pattern for Ss born in the Southern hemisphere to that which has been reported for Northern hemisphere Ss. EPI and birth data were obtained from 450 adult Ss, of whom 338 were New Zealand born. Results for Extraversion significantly matched British findings (p less than .01). Results for Neuroticism were ambiguous. It is concluded that seasonal and climatic influences are unlikely to explain the Extraversion differences between birth groups.

Adult↗