Biomedical subjects

L M Hall

Publications and source records attributed to L M Hall.

At least 37 records · Page 2Linked to original sources

PubMed1999

A study of small-colony, beta-haemolytic, Lancefield group C streptococci within the anginosus group: description of Streptococcus constellatus subsp. pharyngis subsp. nov., associated with the human throat and pharyngitis.

beta-Haemolytic, Lancefield group C streptococci within the anginosus-species group were shown by genetic and phenotypic criteria to be heterogeneous and to constitute two distinct taxa related at subspecies level to Streptococcus constellatus and Streptococcus anginosus, respectively. The first group, referred to here as DNA group 1, comprised six strains with 86-100% intragroup overall genomic DNA relatedness; five of the strains were originally isolated from the human throat and one was from an abdominal mass. They shared 61-77% DNA relatedness (delta Tm values = 1.2-1.5 degrees C) with reference strains of S. constellatus and were clearly differentiated from S. constellatus (now named Streptococcus constellatus subsp. constellatus) by the ability to produce beta-N-acetylgalactosaminidase, beta-N-acetylglucosaminidase, beta-D-fucosidase, beta-D-galactosidase and beta-D-glucosidase. The name S. constellatus subsp. pharyngis is proposed for these strains on the grounds that they are genetically and phenotypically distinct and exhibit a predeliction for the human throat, being isolated also from cases of pharyngitis. The DNA G + C content is 35-37 mol%. The type strain is MM9889aT (= NCTC 13122T). The second group (DNA group 2) was formed by five beta-haemolytic, Lancefield group C strains originally isolated from various human infections. DNA group 2 strains (81-100% intragroup DNA relatedness) shared 60-72% DNA relatedness (delta Tm values = 2.1-4.1 degrees C) with S. anginosus strains NCTC 10713T and MAS 283 but were not clearly differentiated phenotypically from S. anginosus, showed no clear pattern of clinical association, and therefore are not formally proposed as a new subspecies here.

Bacterial Typing Techniques↗

PubMed1999

OXA-17, a further extended-spectrum variant of OXA-10 beta-lactamase, isolated from Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates 871 and 873 were isolated at Hacettepe University Hospital in Ankara and were highly resistant to ceftazidime (MIC, 128 microg/ml). Each produced three beta-lactamases, with pIs of 5.3, 6.1, and 7.9. The beta-lactamase with a pI of 5.3 was previously shown to be PER-1 enzyme. The antibiograms of the isolates were not entirely explained by production of PER-1 enzyme, insofar as ceftazidime resistance was incompletely reversed by clavulanate. The enzymes with pIs of 6.1 and 7.9 were therefore investigated. The enzyme with a pI of 6.1 proved to be a novel mutant of OXA-10, which we designated OXA-17, and had asparagine changed to serine at position 73 of the protein. When cloned into Escherichia coli XL1-blue, OXA-17 enzyme conferred greater resistance to cefotaxime, latamoxef, and cefepime than did OXA-10, but it had only a marginal (two- to fourfold) effect on the MIC of ceftazidime. This behavior contrasted with that of previous OXA-10 mutants, specifically OXA-11, -14, and -16, which predominately compromise ceftazidime. Extracted OXA-17 enzyme had relatively greater activity than OXA-10 against oxacillin, cloxacillin, and cefotaxime but, in terms of kcat/Km, it had lower catalytic efficiency against most beta-lactams. The enzyme with a pI of 7.9 was shown by gene sequencing to be OXA-2.

Anti-Bacterial Agents↗

PubMed1998

Analysis of stable oxidized molecular species of glycerophospholipids following treatment of red blood cell ghosts with t-butylhydroperoxide.

A model of lipid peroxidation was employed to investigate the formation of oxidized phospholipids in red blood cell membranes after treatment with t-butylhydroperoxide (tBuOOH). On-line normal-phase HPLC/mass spectrometry (LC/MS) with electrospray ionization was used to separate phospholipid classes and analyze the distribution of the major poly-unsaturated fatty acyl groups and corresponding oxidation products. Arachidonic acid was observed primarily in plasmalogen glycerophosphoethanolamine (GPE), whereas linoleic acid was equally distributed in 1,2-diacyl-GPE and glycero-phosphocholine (GPC) lipids. The additions of one and two oxygen atoms to poly-unsaturated phospholipid molecular species were observed as the major, stable products after incubation with tBuOOH. Tandem mass spectrometry was utilized to further structurally characterize the oxidized fatty acyl groups which were identified as 5-, 8-, 9-, 11-, 12-, and 15-hydroxy-eicosatetraenoate (HETE) and 5-, 12-, and 15-hydroperoxyeicosatetraenoate (HpETE) in addition to 9- and 13-hydroxyoctadecadienoate (HODE) and 9- and 13-hydroperoxyoctadecadienoate (HpODE). Although 18:0p/20:4-GPE was the predominate phospholipid species containing arachidonic acid, the major species containing HETE and HpETE were the 1,2-diacyl-GPE with hexadecanoate as the sn-1 substituent. This result would be consistent with a differential pathway of oxidative degradation of arachidonoyl plasmalogen GPE suggesting a unique role for this plasmalogen molecular species glycerophospholipid.

Chromatography, High Pressure Liquid↗

PubMed1998

A mutation affecting dihydropyridine-sensitive current levels and activation kinetics in Drosophila muscle and mammalian heart calcium channels.

The Dmca1D gene encodes a Drosophila calcium channel alpha1 subunit. We describe the first functional characterization of a mutation in this gene. This alpha1 subunit mediates the dihydropyridine-sensitive calcium channel current in larval muscle but does not contribute to the amiloride-sensitive current in that tissue. A mutation, which changes a highly conserved Cys to Tyr in transmembrane domain IS1, identifies a residue important for channel function not only in Drosophila muscle but also in mammalian cardiac channels. In both cases, mutations in this Cys residue slow channel activation and reduce expressed currents. Amino acid substitutions at this Cys position in the cardiac alpha1 subunit show that the size of the side chain, rather than its ability to form disulfide bonds, affects channel activation.

Amiloride↗

PubMed1998

Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins.

SmtB from Synechococcus PCC7942 is a trans-acting dimeric repressor that is required for Zn(2+)-responsive expression of the metallothionein SmtA. The structure of SmtB was solved using multiple isomorphous replacement techniques and refined at 2.2 A resolution by simulated annealing to an R-factor of 0.218. SmtB displays the classical helix-turn-helix motif found in many DNA-binding proteins. It has an alpha + beta topology, and the arrangement of the three core helices and the beta hairpin is similar to the HNF-3/fork head, CAP and diphtheria toxin repressor proteins. Although there is no zinc in the crystal structure, analysis of a mercuric acetate derivative suggests a total of four Zn2+ binding sites in the dimer. Two of these putative sites are at the opposite ends of the dimer, while the other two are at the dimer interface and are formed by residues contributed from each monomer. The structure of the dimer is such that simultaneous binding for both recognition helices to DNA would require either a bend in the DNA helix or a conformational change in the dimer. The structure of Synechococcus SmtB is the first in this family of metal-binding DNA repressors.

Amino Acid Sequence↗

PubMed1998

Evolution of the gibbon subgenera inferred from cytochrome b DNA sequence data.

DNA sequences for the mitochondrial cytochrome b gene from the four extant gibbon subgenera are described. The data confirm that the gibbon subgenera evolved from a common hylobatid ancestor and suggest that they diverged from each other after the divergence of the extant African great ape species. The cytochrome b gene does not resolve the evolutionary relationships between the gibbon subgenera themselves.

Animals↗

PubMed1998

Comparison of three methods for the molecular typing of Singapore isolates of enterococci with high-level aminoglycoside resistances.

Enterococci are frequently isolated as nosocomial pathogens and have often acquired intrinsic drug resistances. Molecular typing techniques have been developed to assist in epidemological and infection control measures. This study investigates enterococci with high-level aminoglycoside resistance (HLAR) from the National University Hospital (NUH) of Singapore, and evaluates and compares three methods for typing: restriction enzyme analysis by conventional gel electrophoresis [restriction fragment length polymorphism (RFLP)], pulsed-field gel electrophoresis (PFGE), and polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD). Fifty-two isolates of Enterococcus faecalis and 13 isolates of Enterococcus faecium were used for the study. The numbers of patterns obtained for E. faecalis and E. faecium were 26 and 4, respectively by the RFLP method, and very similar discrimination was obtained by PFGE. RAPD PCR results were not reliably reproducible. A single pattern type by RFLP accounted for 16 of the E. faecalis isolates, suggesting hospital spread.

Aminoglycosides↗

PubMed1998

Electrospray mass spectrometric analysis of 5-hydroperoxy and 5-hydroxyeicosatetraenoic acids generated by lipid peroxidation of red blood cell ghost phospholipids.

Recent evidence suggests that generation of hydroxyl radicals in the presence of lipid membranes can lead to oxidation of arachidonic acid esterified to glycerophospholipids and the production of compounds isomeric to prostaglandins, thromboxanes, and leukotrienes. Liquid chromatography tandem mass spectrometry and multiple reaction monitoring were employed to quantitate the production of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in red blood cells ghosts treated with t-butylhydroperoxide (tBuOOH). Untreated red blood cell ghosts were found to contain low, but measurable quantities of these three 5-oxygenated eicosanoids as phospholipid esters. Following treatment, there was approximately a 53- and 22.5-fold increase in 5-HETE and 5-HPETE, respectively, and an 8.5-fold increase in 5-oxo-ETE. The formation of these compounds was inhibited nearly 90% by the antioxidants butylated hydroxytoluene, ascorbic acid, and resveratrol providing further evidence for free radical mediated oxidation of arachidonic acid. This analytical protocol provided sufficient sensitivity for detection of these compounds in studies in which previous analysis by high-pressure liquid chromatography with UV detection failed to detect their presence. These results reveal that the biologically active eicosanoids 5-HPETE, 5-HPETE, and 5-oxo-ETE are formed esterified to phospholipids following exposure of cellular membranes to reactive oxygen species and free radicals in a model system where intracellular antioxidant mechanisms were depleted.

Chemotactic Factors↗

PubMed1998

Activation of human polymorphonuclear leukocytes by products derived from the peroxidation of human red blood cell membranes.

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)-HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50's of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.

Arachidonic Acids↗

PubMed1998

A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens.

An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identified exons 2-5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum-eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore autosomal. We propose that Ace.x is the recently postulated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance.

Acetylcholinesterase↗

PubMed1998

Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.

Alleles↗

PubMed1998

Can susceptibility to an antimicrobial be restored by halting its use? The case of streptomycin versus Enterobacteriaceae.

To test the widespread view that resistance disappears in the absence of antimicrobial use, we tested streptomycin against 477 Enterobacteriaceae from the Royal London Hospital. Twenty per cent proved resistant although streptomycin is little used at the hospital and streptomycin resistance in gram-negative bacteria is caused by mechanisms that do not compromise the drugs that are used. Up to 70% of the observed resistance was associated with cross-resistance to spectinomycin and the presence of ant(3")-Ia, an integron-associated gene carried in Tn21-type transposons. This genetic organization may have conserved streptomycin resistance in the absence of direct selection pressure.

Anti-Bacterial Agents↗

PubMed1998

Epidemiological typing of klebsiellae with extended-spectrum beta-lactamases from European intensive care units.

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to oxyimino-aminothiazolyl cephalosporins, especially in klebsiellae. In a recent survey we detected ESBLs in 220 (23%) of 966 consecutive klebsiellae from 35 intensive care units (ICUs) in southern and western Europe. The present study examined the extent to which this distribution reflected epidemic strain spread, as against the distribution of ESBL genes into unrelated strains. All 220 ESBL producers were subjected to capsular serotyping and pulsed-field gel DNA electrophoresis (PFGE). Beta-Lactamases were typed for strains isolated on three or more occasions, with the emphasis on SHV enzymes, as these were commoner than TEM variants. Serotyping and PFGE typing defined 85 distinct strains, from 23 of the 35 participating centres. Of 14 centres that contributed five or more ESBL producers, all sent representatives of more than one strain, and two centres sent members of ten or more different strains in contributions of 17-21 ESBL-producing isolates. Nevertheless, epidemic strains-defined as those represented by three or more isolates-accounted for a majority (61%) of the collection. Fifty-two isolates of the same serotype K25 (occasionally acapsular) strain with SHV-4 beta-lactamase were recovered at two French hospitals and one in Belgium. This strain has been found by others in France, and has become particularly widespread. Another single strain was found in two separate Portuguese centres, and many individual hospitals had one or more epidemic strain(s), as well as a scatter of diverse ESBL producers. Major variation in antibiogram and plasmid profile was apparent within strains, with some intra-strain variation in beta-lactamase type. These data imply a fluid situation, with resistance determinants being gained, modified or lost. The endemicity of ESBL producers is disturbing since it limits the potential for control by blocking strain spread, while the diversity within strains is disturbing because it complicates the design of antibiotic policies even during 'single strain' outbreaks.

Anti-Bacterial Agents↗

PubMed1998

OXA-16, a further extended-spectrum variant of OXA-10 beta-lactamase, from two Pseudomonas aeruginosa isolates.

Two extended-spectrum mutants of the class D beta-lactamase OXA-10 (PSE-2) from Pseudomonas aeruginosa isolates obtained in Ankara, Turkey, were described previously and were designated OXA-11 and -14. P. aeruginosa 906 and 961, isolated at the same hospital, were highly resistant to ceftazidime (MIC >/= 128 microgram/ml) and produced a beta-lactamase with a pI of 6.2. The MICs of ceftriaxone, cefoperazone, cefsulodin, and cefepime were 4- to 16-fold above the typical values for P. aeruginosa, whereas the MICs of penicillins and cefotaxime were raised only marginally. Ceftazidime MICs were not significantly reduced by clavulanate or tazobactam at 4 microgram/ml. Ceftazidime resistance did not transfer conjugatively but was mobilized to P. aeruginosa PU21 by plasmid pUZ8. Both isolates gave similar DNA restriction patterns, suggesting that they were replicates; moreover, they yielded identically sized BamHI fragments that hybridized with a blaOXA-10 probe. DNA sequencing revealed that both isolates had the same new beta-lactamase, designated OXA-16, which differed from OXA-10 in having threonine instead of alanine at position 124 and aspartate instead of glycine at position 157. The latter change is also present in OXA-11 and -14 and seems critical to ceftazidime resistance. Kinetic parameters showed that OXA-16 enzyme was very active against penicillins, cephaloridine, cefotaxime, and ceftriaxone, but hydrolysis of ceftazidime was not detected despite the ability of the enzyme to confer resistance.

Amino Acid Sequence↗

PubMed1998

Conservation of restriction sites in isolates of Streptococcus pneumoniae with diverse restriction fragment patterns.

Separation of large restriction fragments by pulsed-field gel electrophoresis is a commonly used method for epidemiological typing of Streptococcus pneumoniae and many other bacterial species. Information on the genetic changes underlying the restriction fragment polymorphisms that allow discrimination between isolates is scarce. In this study fragments adjacent to ApaI sites in a clinical isolate of S. pneumoniae were cloned and used to probe HindIII and HindIII-plus-ApaI genomic DNA digests from other isolates with very different ApaI fragment patterns. If for a given isolate the HindIII fragment detected by the probe was reduced in size on digestion with ApaI, it was deduced that the ApaI site was conserved in that isolate. The results demonstrate that of six ApaI sites in PN93/908 examined, five were retained in 11 genetically different isolates and one was retained in 2 isolates but lost in 9 others. It was concluded that point mutations at restriction sites are unlikely to account for the restriction fragment length polymorphism observed and that much of the polymorphism may be due to DNA rearrangements, possibly resulting from the insertion or deletion of mobile DNA elements.

DNA Restriction Enzymes↗

PubMed1998

Application of molecular typing to the epidemiology of Streptococcus pneumoniae.

The spread of antibiotic resistance and the development of new vaccines have focused attention on the epidemiology of Streptococcus pneumoniae over recent years. While serotyping and the determination of antibiotic resistance remain primary methods for characterising pneumococci, molecular typing can add greater discrimination and complementary information. Methods based on restriction fragment length polymorphism within total DNA or non-specific polymerase chain reaction provide information representative of the whole genome and can be used to recognise closely related isolates from different sources, whether in the investigation of possible cross infection at the local level or in the investigation of national or international spread of antibiotic resistant strains. Fingerprinting of penicillin binding protein genes adds further information in the analysis of penicillin resistant isolates. The use of a combination of typing methods to analyse both the genome as a whole and specific loci has led to the realisation that pneumococci undergo horizontal gene transfer much more often than most other bacterial species. In particular the spread of penicillin resistance has been characterised by a combination of the spread of epidemic strains, transfer of chromosomal resistance genes from such strains into other genetic backgrounds, and transfer of capsule genes resulting in the switch of serotypes within strains. In the future molecular typing will have an important role in discovering whether widespread vaccination leads to genetic modification of the pneumococcal population causing invasive disease.

Bacterial Proteins↗

PubMed1997

The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn2+ ions per subunit.

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

Bacterial Proteins↗

PubMed1997

Functional expression and characterization of skeletal muscle dihydropyridine receptors in Xenopus oocytes.

Dihydropyridine receptors in vertebrate skeletal muscle serve a dual role: as voltage sensors for excitation-contraction coupling and as voltage-activated calcium channels. Although they were the first of six classes of calcium channels to be cloned, skeletal muscle dihydropyridine receptors remain the only ones not functionally expressed as calcium channels in Xenopus oocytes, leading to the hypothesis that an interacting component is missing. Using beta1b, an isoform previously found in brain, we have for the first time reconstituted skeletal muscle calcium channel function in Xenopus oocytes. We show that this beta subunit is necessary for functional expression and that the alpha2delta subunit significantly enhances the expressed current. The majority of the alpha1 subunit in skeletal muscle is a truncated form. Here we show that both the full-length and truncated forms produce functional calcium channels in Xenopus oocytes, but the truncated form gives significantly larger currents. In addition, we show that the beta1b transcript is expressed in rat skeletal muscle, although at a much lower level than the abundant beta1a isoform.

Animals↗