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A J Lax

Publications and source records attributed to A J Lax.

At least 37 records · Page 2Linked to original sources
PubMed1995

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH.

The susceptibility of the potent mitogen Pasteurella multocida toxin (PMT) to various proteases was investigated. PMT at a toxin to protease molar ratio of 1:1 was resistant to 8 of the 11 proteases tested after one hour. With longer incubation, PMT remained resistant to 7 proteases, and this correlated with a retention of biological activity, indicating that PMT might not require proteolytic cleavage at least until it bound to a cell receptor. Previous evidence had suggested that PMT is processed in the cell via an endosome or lysosome. We have shown that PMT became susceptible to proteolysis when the pH was lowered to 5 or below. This supports the previous suggestion that PMT is processed via a low pH compartment in the cell.

3T3 Cells↗
PubMed1994

Mutation of a putative ADP-ribosylation motif in the Pasteurella multocida toxin does not affect mitogenic activity.

Pasteurella multocida toxin (PMT) is a potent mitogen for Swiss 3T3 fibroblasts and cytotoxic to embryonic bovine lung cells. Site-directed mutagenesis was used to investigate the functional significance of a three amino acid motif in PMT that is present in five other bacterial protein toxins which exhibit ADP-ribosyl transferase activity. Crude lysates of mutant clones were fully cytotoxic for embryonic bovine lung cells. Purified mutant toxin was also as effective at stimulating inositol phosphate turnover and nucleic acid synthesis as wild type toxin. We conclude that this motif has no functional significance in Pasteurella multocida toxin.

3T3 Cells↗
PubMed1994

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes.

The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using promoter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present within the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.

Animals↗
PubMed1994

Development of a probe and PCR primers specific to the virulence plasmid of Salmonella enteritidis.

Restriction analysis of the Salmonella enteritidis virulence plasmid, followed by hybridisation with radiolabelled S. typhimurium and S. dublin plasmids, revealed a 2-kb Pstl/Bg/l fragment that was specific to S. enteritidis. Colony hybridisation experiments with this fragment detected 29 out of 31 S. enteritidis strains tested. S. blegdam, S. moscow and S. paratyphi C also hybridised with this fragment and a comparison of the plasmids from these serotypes revealed striking similarities. A Pstl/Pvull sub-clone of the 2-kb fragment was used to design primers for quantitative polymerase chain reaction (QPCR) detection of S. enteritidis from broth culture.

Base Sequence↗
PubMed1994

Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.

The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.

Animals↗
PubMed1993

A quantitative polymerase chain reaction method for the detection in avian faeces of salmonellas carrying the spvR gene.

A quick, semi-quantitative method of detecting Salmonella species which contain the virulence plasmid has been developed using the polymerase chain reaction (PCR). A pair of primers have been synthesized encompassing a 500 bp fragment of the spvR virulence gene. Competitor DNA consisting of the spvR gene with a 94 bp deletion situated between the primer recognition sequences, was cloned into a plasmid vector. Co-amplification of the 'unknown' target salmonella DNA with known quantities of competitor DNA in the same reaction tube gave PCR products of 500 and 406 bp respectively. Visual assessment of the ratio of the two products on ethidium bromide stained agarose gels provided an estimate of the approximate number of salmonella cells present in avian faeces. The technique could be applied to detect quantifiably any non-host DNA in clinical samples if a suitable DNA sequence for primer construction is available.

Animals↗
PubMed1993

Molecular analysis of spv virulence genes of the Salmonella virulence plasmids.

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium, Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon-poor media, and optimal expression is dependent on the katF locus. The virulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Gene Expression Regulation, Bacterial↗
PubMed1992

Pasteurella multocida toxin is a potent inducer of anchorage-independent cell growth.

The growth of many normal cells requires contact with an adhesive substratum, a requirement that is frequently abrogated in the transformed phenotype. We have explored pathways that can lead to the anchorage-independent growth of cultured Rat-1 fibroblasts. Pasteurella multocida toxin (PMT), a 146-kDa mitogenic protein, caused a striking increase in the formation of colonies (greater than 200 microns) from single cells in soft agar. The magnitude of the effect of PMT was greater than that achieved by epidermal growth factor or platelet-derived growth factor. The toxin was extremely potent, with half-maximal and maximal effects observed at 1 and 10 pM PMT, respectively. This concentration dependence of the action of the toxin is similar to that for the stimulation of DNA synthesis in adherent cultures of the cells. Stimulation of colony formation could be achieved by a transient exposure of the cells to PMT and it was blocked by methylamine, indicating that the toxin enters the cells to act. Colony formation was stimulated equally by native and recombinant PMT, but a truncated version (33.5 kDa) of the recombinant toxin was ineffective. PMT antiserum blocked colony formation in response to PMT. In the Rat-1 cells, PMT stimulated the phospholipase C-mediated hydrolysis of inositolphospholipids, as indicated by the stimulation of inositol phosphate release, Ca2+ mobilization, and phosphorylation of a protein kinase C substrate. The results indicate that the deregulation of signal-transduction pathways as elicited by an intracellularly acting bacterial toxin can induce a malignant phenotype.

Animals↗
PubMed1992

A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient-limited conditions.

This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal-medium containing various sole carbon/energy sources, unlike the wild-type and plasmid-cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the phenotypic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal-medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.

Alleles↗
PubMed1991

Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.

Pasteurella multocida toxin, both native and recombinant, is an extremely potent mitogen for Swiss 3T3 cells and acts to enhance the formation of total inositol phosphates (Rozengurt, E., Higgins, T., Changer, N., Lax, A.J., and Staddon, J.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 123-127). P. multocida toxin also stimulates diacylglycerol production and activates protein kinase C (Staddon, J.M., Chanter, N., Lax, A.J., Higgins, T.E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848). Here we analyze, by [3H]inositol labeling and high performance liquid chromatography, the inositol phosphates in recombinant P. multocida toxin-treated cells. Recombinant P. multocida toxin stimulated increases in [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and its metabolic products, including Ins(1,3,4,5)P4, Ins(1,3,4)P3, Ins(1,4)P2, Ins(4/5)P, and Ins(1/3)P. The profile of the increase in the cellular content of these distinct inositol phosphates was very similar to that elicited by bombesin. Furthermore, recombinant P. multocida toxin, like bombesin, mobilizes an intracellular pool of Ca2+. Recombinant P. multocida toxin pretreatment greatly reduces the Ca2(+)-mobilizing action of bombesin, consistent with Ca2+ mobilization from a common pool by the two agents. The enhancement of inositol phosphates and mobilization of Ca2+ by recombinant P. multocida toxin were blocked by the lysosomotrophic agents methylamine, ammonium chloride, and chloroquine and occurred after a dose-dependent lag period. The stimulation of inositol phosphate production by recombinant P. multocida toxin persisted after removal of extracellular toxin, in contrast to the reversibility of the action of bombesin. Recombinant P. multocida toxin, unlike bombesin and guanosine 5'-O-(gamma-thiotriphosphate), did not cause the release of inositol phosphates in permeabilized cells. These data demonstrate that recombinant P. multocida toxin, acting intracellularly, stimulates the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Ammonium Chloride↗
PubMed1990

Sequence analysis of the potent mitogenic toxin of Pasteurella multocida.

Pasteurella multocida toxin is a potent mitogen for cultured Swiss 3T3 cells where it causes an accumulation of inositol phosphates and activation of protein kinase C. The gene sequence described here coded for a 146 kDa protein. The ORF was preceded by a ribosome binding site and followed by a stem loop. There was no evidence for a signal sequence. The gene had a low G + C base ratio which differs from the rest of the Pasteurella genome. There was no significant homology with other known proteins, although a motif found in certain bacterial toxins which are ADP-ribosyl transferases is present. A recombinant expressing only part of the PMT gene was not mitogenic.

Amino Acid Sequence↗
PubMed1990

Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.

Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.

Animals↗
PubMed1990

Identification of proteins expressed by the essential virulence region of the Salmonella dublin plasmid.

An 8 kilobase pair (kb) fragment from the Salmonella dublin 2229 plasmid is sufficient to restore virulence for mice to a cured strain of S. dublin. Deletion analysis of this virulence fragment identified at least one specific region required for virulence expression. Plasmid-directed protein synthesis in minicells has indicated the presence of at least four genes within the essential virulence region of the S. dublin plasmid, encoding proteins of 70, 33, 30 and 26 kDa. Analysis of the proteins expressed by the deletion derivatives suggested that expression of the 33 kDa polypeptide was linked to that of the 30 kDa polypeptide. The proteins expressed by the essential virulence region of the S. dublin plasmid appeared to be similar to the plasmid-encoded virulence proteins recently identified in S. typhimurium.

Animals↗
PubMed1990

Pasteurella multocida toxin: potent mitogen for cultured fibroblasts.

Native Pasteurella multocida toxin (PMT) is shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37 degrees C, but not at 4 degrees C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.

Animals↗
PubMed1990

Cloning of the toxin gene from Pasteurella multocida and its role in atrophic rhinitis.

The gene for the osteolytic toxin of Pasteurella multocida has been cloned into a plasmid vector and expressed off its own promoter in Escherichia coli. Particular restriction endonucleases failed to cut the gene and regions flanking it, suggesting an A + T base ratio significantly greater than the remaining genome of P. multocida. Cloned toxin was indistinguishable from the native toxin with respect to molecular mass, antigenicity and toxicity in different tests. A single intraperitoneal injection of toxin purified from the recombinant E. coli reproduced in gnotobiotic pigs the pathological changes characteristic of atrophic rhinitis. The recombinant E. coli produced at least 10 times as much toxin as P. multocida.

Animals↗
PubMed1990

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis.

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.

Blotting, Southern↗