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P M Bennett

Publications and source records attributed to P M Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1998

Structural changes in samples cryofixed by contact with a cold metal block.

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.

Animals↗
PubMed1998

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria.

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.

Aeromonas↗
PubMed1997

Entanglement in fishing gear and other causes of death in cetaceans stranded on the coasts of England and Wales.

Between August 1990 and September 1995 the carcases of 422 cetaceans of 12 species that had died around the coasts of England and Wales were examined. There were 234 harbour porpoises (Phocoena phocoena), 138 common dolphins (Delphinus delphis), and 50 individuals of 10 other species of dolphins and whales. The cause of death was diagnosed in the harbour porpoises and common dolphins was entanglement in fishing gear (bycatch). Of the cases in which the cause of death was established, 66 (38 per cent) of 176 harbour porpoises, 86 (80 percent) of 108 common dolphins, and six (19 per cent) of 31 individuals of other species had been bycaught. Neonatal starvation, pneumonia and generalised infections accounted for a further 31 per cent of the diagnosed causes of death in harbour porpoises. The proportion of stranded common dolphins that had been bycaught was consistently high except during 1995, but the proportion of stranded harbour porpoises which had been bycaught increased in each successive year.

Animals↗
PubMed1997

Evidence that the tandem Ig domains near the end of the muscle thick filament form an inelastic part of the I-band titin.

In vertebrate striated muscle the titin/connectin molecule spans half a sarcomere, and in the I-band forms and elastic filament connecting the Z-line with the end of the thick filaments. The only part of the elastic filament that has been described in intact rest length muscle is a short extension to the thick filament observed in freeze-fractured cardiac muscle which has similarities to the end-filament of negatively stained isolated thick filaments. We report here further observations made in sections of rabbit psoas muscle. In very thin longitudinal sections thin extensions to the thick filaments some 0.11 micron long and 5-6 nm in diameter are seen. Transverse sections show that each thick filament has such an extension. Nothing similar is seen further into the I-band or at the Z-line. The common features of this structure in both cardiac and skeletal muscle suggest that it corresponds to a common sequence in their titins. Such a sequence is to be found in the 22 tandem Ig domains near the A/I junction. Taken together with other information about the arrangement of domains in this part of the sarcomere, this leads to a calculated length for the end extension of 104 nm. The length of the extension does not vary with sarcomere length between 2.2 and 3.0 microns and therefore it corresponds to an inelastic region of I-band titin over the physiological range. Each extension probably comprises part of three to six titin molecules depending on the complement of titin in the thick filament, as previously suggested. A polymer formed from several strands of Ig domains would make for a relatively rigid structure which would resist folding or stretching when subjected to the small passive forces which pertain over the physiological range of sarcomere lengths. The relationship of the N2-line with the end-filament has also been studied. The N2-line position varies with sarcomere length in an elastic manner. Only at short sarcomere lengths does the end of the end-filament coincide with the position of the N2-line. Taking into account recent work on the elasticity of titin in the I-band we conclude that the N2-line corresponds to part of the elastic PEVK region of titin and not the region of titin sequence designated N2.

Actin Cytoskeleton↗
PubMed1997

Distribution and expression of beta-lactamase genes among Aeromonas spp.

Clinical and environmental isolates of Aeromonas spp. (20 Aeromonas caviae, 33 Aeromonas veronii and 21 Aeromonas hydrophila) were examined for their ability to yield mutants derepressed for beta-lactamase production and for the distribution of the three chromosomally encoded beta-lactamase genes, ampS and cepS from A. veronii bv. sobria and cphA from A. hydrophila. Of these isolates, 100% and 96% of the isolates from A. hydrophila and A. caviae, respectively, yielded beta-lactamase derepressed mutants but only 38% of A. veronii isolates yielded the resistant phenotype at 37 degrees C. However, when tested at 30 degrees C, all isolates gave rise to the derepressed mutants, indicating a temperature effect on the control mechanism. All mutants had significantly higher beta-lactamase activity against ampicillin, oxacillin, cephaloridine and imipenem. Hybridization studies with cloned aeromonas beta-lactamase genes indicated that the cephalosporinase gene, cepS, is almost ubiquitous for the three species tested. The cphA gene cross-hybridized with all isolates of A. veronii and A. hydrophila but not to A. caviae isolates. In contrast, hybridization studies using ampS revealed that only 25% of A. caviae, 45% of A. veronii and 38% of A. hydrophila tested carried the ampS gene or one closely homologous to it. Nonetheless, strains that failed to hybridize with ampS showed two serine beta-lactamases when analysed by isoelectric focusing.

Aeromonas↗
PubMed1997

Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

Amino Acid Sequence↗
PubMed1996

Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria).

In 1992 we began an investigation into incidents of unusual and mass mortalities of the common frog (Rana temporaria) in Britain which were being reported unsolicited to us in increasing numbers by members of the public. Investigations conducted at ten sites of unusual mortality resulted in two main disease syndromes being found: one characterized by skin ulceration and one characterized by systemic haemorrhages. However, frogs also were found with lesions common to both of these syndromes and microscopic skin lesions common to both syndromes were seen. The bacterium Aeromonas hydrophila, which has been described previously as causing similar lesions, was isolated significantly more frequently from haemorrhagic frogs than from those with skin ulceration only. However, as many of the latter were euthanased, this may have been due to differences in post mortem bacterial invasion. An iridovirus-like particle has been identified on electron microscopical examination of skin lesions from frogs with each syndrome and iridovirus-like inclusions have been detected in the livers of frogs with systemic haemorrhages. Also, an adenovirus-like particle has been cultured from one haemorrhagic frog. A poxvirus-like particle described previously from diseased frogs has now been found also in control animals and has been identified as a melanosome. Both the prevalence of the iridovirus-like particle and its association with lesions indicate that it may be implicated in the aetiology of the disease syndromes observed. Specifically, we hypothesize that primary iridovirus infection, with or without secondary infection with opportunistic pathogens such as A. hydrophila, may cause natural outbreaks of 'red-leg', a disease considered previously to be due to bacterial infection only.

Adenoviridae↗
PubMed1996

Titin domain patterns correlate with the axial disposition of myosin at the end of the thick filament.

Titin has been suggested to act as a molecular ruler for the precise assembly of thick filaments in vertebrate striated muscle. To investigate the correlation of titin domain patterns with the architecture of the thick filament at its end, we have investigated the axial position of titin epitopes at the thick-filament/I-band junction. Antibodies against immunoglobin (Ig) domains N and C-terminal to the unique block of six fibronectin-3 (fn3) domains in this region were used. The distance between these epitopes confirms the idea that titin is laid out linearly along the thick filament with each domain measuring about 4 nm in length. Our data demonstrate that the gap of myosin crossbridges near the end of the thick filament closely correlates with the stretch of six fn3 domains, and that the last two crossbridges are at the level of the first two groups of fn3 domains which were previously assigned to the I-band. We conclude that the pattern of groups of fn3 domains reflects the arrangement of the myosin heads, at least at the end of the A-band. It seems likely that an alteration in the interaction between myosin and the titin fn3 domains towards the end of the thick filament is important for the formation of the crossbridge gap and thus the termination of the thick filament.

Actin Cytoskeleton↗
PubMed1996

Adverse drug reactions in a hospital general medical unit meriting notification to the Committee on Safety of Medicines.

1. We have retrospectively analysed data collected by a local adverse drug reactions reporting scheme in an acute hospital medical setting and have determined the numbers and types of reactions that would have merited notification as yellow card reports according to the guidelines of the Committee on Safety of Medicines. 2. The data related to 20,695 consecutive acute general medical admissions on seven general medical wards (140 beds) and were collected over 3 years, from April 1990 to March 1993. 3. Over 3 years there were 1420 reports of suspected adverse drug reactions, a rate of 68.7 per 1000 admissions. 4. If the guidelines for reporting issued by the Committee on Safety of Medicines had been strictly followed, 477 yellow cards would have been sent (23.1 per 1000 admissions). In 357 of these reports (74.8%), the reaction had caused admission to hospital. Only 31 of the 477 potential cards (6.5%) involved black triangle drugs and 10 of these were for minor reactions. 5. Only 30 of the 477 potential yellow cards (6.3%) were known to have been sent. The majority of those reactions not reported were for drug-related admissions, most of which were for well-known reactions to established drugs. 6. We have confirmed and quantified the extent of under-reporting of serious suspected adverse drug reactions to the Committee on Safety of Medicines from our hospital medical unit.

Adverse Drug Reaction Reporting Systems↗
PubMed1996

Enzyme kinetics and biochemical analysis of ImiS, the metallo-beta-lactamase from Aeromonas sobria 163a.

The metallo-beta-lactamase from Aeromonas sobria 163a, ImiS, was isolated in a two stage purification procedure using protein affinity columns. Enzyme kinetics show that ImiS hydrolyses the carbapenems but displays poor activity against other beta-lactams. ImiS possesses the narrowest spectrum of activity of the Group 3 enzymes that have been analysed. Sequencing of the 40 N-terminal amino acids show this region to be identical to that of the CphA metallo-beta-lactamase from Aeromonas hydrophila (Massidda, Rossolini & Satta, 1991). Light scattering analysis indicates that ImiS is functionally active as a monomer.

Aeromonas↗
PubMed1995

Concentrated Tris solutions for the preparation, depolymerization, and assay of actin: application to erythroid actin.

High concentrations of Tris are effective in dissociating actin-containing complexes, such as the red cell membrane cytoskeleton. A preparative procedure for red cell actin is based on the dissociation of the membrane skeletal complex in a buffer containing 1 M Tris hydrochloride, followed by gel filtration chromatography in the same medium. The actin is recovered as the monomer and is fully native, as judged by its critical concentration of polymerization, inhibition of DNase I, stimulation of myosin ATPase, and the appearance in the electron microscope of filaments, both bare and decorated with heavy meromyosin, and of magnesium ion-induced paracrystals. The Tris solution causes rapid depolymerization of F-actin with no denaturation, and the solution of monomeric actin in this medium is stable for many weeks in the cold; concentrated Tris is more reliable than guanidinium chloride for the depolymerization of F-actin in the estimation of total actin concentration by the DNase I inhibition assay.

Actins↗
PubMed1995

Evaluation of freeze substitution in rabbit skeletal muscle. Comparison of electron microscopy to X-ray diffraction.

Rabbit psoas muscle fibres, relaxed and in rigor, have been freeze substituted for electron microscopy. Fourier transforms and average density maps of micrographs of transverse sections have been obtained and compared to X-ray diffraction data. The Fourier amplitudes from rigor and relaxed muscle are comparable to equatorial data from X-ray diffraction of muscle if there is more disorder in the electron micrographs which can be described by a 'temperature' factor. The phases of reflections out to the 3,2 have been determined; those reflections at the same radius and therefore not separable in the X-ray patterns, such as the 2,1 and the 1,2, are separated in the transforms of sections through the A band. In transforms from both rigor and relaxed muscle they have the same phase. In rigor muscle they have different amplitudes. All the phases are positive or negative showing that the lattice is centrosymmetric at the resolution obtained. The phases obtained generally support those suggested by model building studies using X-ray diffraction data. In rigor muscle, areas where the cross-bridges are regularly attached are clearly seen in thin transverse sections. A handedness to this structure is indicated by a lack of mirror symmetry, in both the Fourier transform of thick sections, and in the averaged density map. This correlates well with the arrangement where the myosin head is bound as in the acto-S1 structure but only to actin monomers within a limited azimuthal range.

Animals↗
PubMed1995

Typing of Listeria monocytogenes by random amplified polymorphic DNA (RAPD) analysis.

The aim of the study was to determine the effectiveness of random amplified polymorphic DNA analysis in typing Listeria monocytogenes from human infections. Twenty-five L. monocytogenes serogroup 1/2 and 70 serogroup 4 including 14 serovar 4b(x) were typed by RAPD-PCR analysis. Six primers were used to type each L. monocytogenes isolate and the DNA amplification performed with supertaq DNA polymerase in a Hybaid Thermal Reactor. Each bacterial strain was analysed separately with all primers and the profiles were judged by eye and designated to a group by comparison to other strains. Bands were classified as major or minor. Based on analysis of major band patterns, the 25 serogroup 1/2 isolates gave rise to 12 different groups. The groups only contained serovar 1/2a or 1/2b with a single exception. Using minor bands all isolates could be distinguished. All the serogroup 4 isolates gave the same major band patterns. The 14 serovar 4b(x) isolates which were epidemiologically related gave identical profiles with the exception of one isolate. Of the remaining strains, 41 produced individual patterns on minor band analysis. RAPD analysis with multiple primers is low cost, discriminatory and is most ideally suited to testing small (< 50) numbers of strains. We have shown that serogroup 1/2 L. monocytogenes strains are a more diverse group than serovar 4b strains and RAPD-PCR will provide a technique of considerable value in typing L. monocytogenes in the future.

Bacteriophage Typing↗
PubMed1995

Inducible expression of the chromosomal cdiA from Citrobacter diversus NF85, encoding an ambler class A beta-lactamase, is under similar genetic control to the chromosomal ampC, encoding an ambler class C enzyme, from Citrobacter freundii OS60.

This study aimed to characterize the molecular basis of beta-lactamase induction in Citrobacter diversus. The chromosomal beta-lactamase encoding region from C. diversus, strain NF85, was cloned and expressed in Escherichia coli. The cloned region was sequenced and open-reading frames encoding a class A beta-lactamase, designated cdiA, and a putative LysR-type transcriptional regulator protein, divergently transcribed from the beta-lactamase gene and designated cdiR, were identified. The nucleotide sequence of the NF85 cdiA was identical to that of the published C. diversus ULA27 ampC sequence. A putative helix-turn-helix DNA-binding motif was located at the N-terminus of CdiR, and homology with enterobacterial AmpR proteins was noted. CdiR was demonstrated to bind to the C. diversus cdiAR intergenic region but not to the C. freundii ampCR intergenic region. A putative CdiR binding motif was identified in the cdiAR intergenic region. The cloned cdiAR region was inducible in E. coli strains SNO3 and HfrH. The inducible phenotype was dependent on the E. coli ampD and ampG gene products. We conclude that the molecular basis of inducible cdiA expression in C. diversus is similar to that of C. freundii ampC.

Amino Acid Sequence↗
PubMed1995

A clinical isolate of Aeromonas sobria with three chromosomally mediated inducible beta-lactamases: a cephalosporinase, a penicillinase and a third enzyme, displaying carbapenemase activity.

Hydrolytic profiles of Aeromonas sobria 163a, non-induced and induced, and of a derepressed mutant 163a-M, indicate that this strain has inducible beta-lactamases with activities against cephalosporins, penicillins and carbapenems. Three enzymes were identified and two of the beta-lactamase genes, ampS, encoding a penicillinase and cepS, encoding a cephalosporinase, were cloned into Escherichia coli, permitting analysis of the individual enzymes. Isoelectric focusing (IEF) analysis using inhibition profiles with EDTA and BRL 42715, confirmed AmpS (pI 7.9) and CepS (7.0) to be serine beta-lactamases. A third beta-lactamase displaying hydrolytic activity against the carbapenems was inhibited by EDTA. The carbapenemase had a pI of 9.3 and was detected on IEF gels by overlaying the gel with agarose containing imipenem and the chromogenic pH indicator, bromothymol blue. Co-inducibility and the recovery of a derepressed mutant in which all three enzymes were produced at high levels indicate that this isolate of A. sobria has three co-ordinately controlled beta-lactamase genes.

Aeromonas↗
PubMed1995

Sequence analysis of two chromosomally mediated inducible beta-lactamases from Aeromonas sobria, strain 163a, one a class D penicillinase, the other an AmpC cephalosporinase.

Two beta-lactamase genes from Aeromonas sobria, strain 163a, have been cloned and sequenced, one encoding a typical class C cephalosporinase, designated CepS, the other a class D penicillinase, designated AmpS. CepS is predicted to be a mature protein of 38 kDa with a pI value of 7.0. The amino acid sequence of CepS is most similar to that of AmpC from Pseudomonas aeruginosa (54.7%). AmpS is predicted to be a mature protein of 27 kDa with a pI value of 7.9 that mostly closely resembles BLAD from Klebsiella pneumoniae (42.2%), and OXA-1 from Escherichia coli (36.6%), beta-lactamases that are encoded by genes carried on multiresistant transposons. AmpS differs significantly from the other class D beta-lactamases in that it hydrolyses cloxacillin poorly and is inducible. Both genes exhibit a high overall GC content and possess a high NNC and NNG codon preference, similar to that of genes from Pseudomonas spp.

Aeromonas↗
PubMed1995

Intestinal ammonium transport by ammonium and hydrogen exchange.

BACKGROUND: Ionized ammonia (NH3) transport in the intestine has not been previously established as a mechanism of acidosis in urinary intestinal diversion or hepatic failure. STUDY DESIGN: The purpose of this study was to establish that ionized transport of ammonium (NH4) occurs in the intestine and to characterize the mechanism of its transport using the methodology of brush border membrane vesicles and acridine orange fluorescence. RESULTS: An NH4/H exchange was demonstrated and found to be the dominant mechanism causing a pH change when NH4 is transported across the lumenal membrane. Ionized NH4 transport was demonstrated to occur against an NH3 concentration gradient. The Km was 1.02 mmol and the Vmax was 247 U/sec. The Hill coefficient was 0.97, indicating a single port. Ammonium hydrogen exchange could be inhibited by amiloride but not by bumetanide. Sodium, potassium or chloride, or both, did not effect the NH4/H exchanger. CONCLUSIONS: This study establishes that ionized NH4 transport occurs across the small intestine brush border in exchange for a hydrogen ion.

Acridine Orange↗