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Biomedical subjects

P A Lambert

Publications and source records attributed to P A Lambert.

At least 19 recordsLinked to original sources
PubMed1992

Enzyme-linked immunosorbent assay for the detection of antibodies to exocellular proteins of Staphylococcus aureus in bone infection.

An enzyme-linked immunosorbent assay (ELISA) was developed for measurement of the antibody response to exocellular protein antigens of Staphylococcus aureus. The wells of a microtitre plate were coated with the exocellular proteins present in the tryptic soya broth growth medium from a stationary phase culture of a bone infection strain of S. aureus. The wells were then reacted sequentially with patient sera, protein A-peroxidase conjugate and chromogenic substrate. Serum from patients with S. aureus bone infection gave a significantly higher IgG titre than sera from patients with Staphylococcus epidermidis or Streptococcus sanguis bone infection or healthy uninfected individuals. The assay therefore appears to have potential in the serodiagnosis of S. aureus bone infection.

Antibodies, Bacterial↗
PubMed1992

Structural studies on bio-active compounds. 20. Molecular modeling and crystallographic studies on methylbenzoprim, a potent inhibitor of dihydrofolate reductase.

Methylbenzoprim (MBP) is a potent inhibitor of dihydrofolate reductase, which is more selective for mammalian than bacterial enzymes. Crystal-structure studies on the free base of MBP, with two independent molecules, and the ethanesulfonate salt, have demonstrated three significantly different conformations for MBP. With the MOPAC optimized MBP cation as starting point, the COSMIC energy was monitored as torsion angles were changed in 5 degrees increments. The barrier to rotation about C(5)-C(11) can create two slowly interconverting rotamers, in agreement with NMR studies. Two conformations of the cation that fit the human DHFR structure from the Brookhaven Protein Data Bank have been found. They differ chiefly by a half-turn about C(5)-C(11), positioning the nitro group on opposite sides but allowing the central and benzylic rings to find hydrophobic surroundings. The central ring is close enough to the predicted position of the cofactor NADPH to make competition likely. Kinetic studies with rat liver DHFR show that MBP is an inhibitor that competes with NADPH as well as dihydrofolate.

Binding Sites↗
PubMed1992

Extracellular proteins as a potential marker of active Staphylococcus aureus infection in bone.

The potential of extracellular protein antigens of Staphylococcus aureus as markers of infection of bone was investigated by immunoblotting. Serum from patients with S. aureus bone infection showed levels of IgG antibodies to a preparation of soluble extracellular proteins of 17-81 kDa which were significantly higher than those found in serum from normal controls. By contrast, immunoblots of various whole cell or cell wall-derived antigens gave complex patterns of response which were unsuitable for positive diagnosis of S. aureus bone infection.

Antibodies, Bacterial↗
PubMed1992

High-level beta-lactamase activity in sputum samples from cystic fibrosis patients during antipseudomonal treatment.

The in vivo activity and source of beta-lactamase in sputum samples from 43 patients with cystic fibrosis (CF) during a 2-week antipseudomonal treatment were studied. A colorimetric method, based on the conversion of nitrocefin, was used for quantitation of the sputum beta-lactamase activity. beta-Lactamases in sputum were characterized by isoelectric focusing and inhibition profile and were compared with the beta-lactamases extracted from Pseudomonas aeruginosa isolated from the paired sputum samples. We found that the beta-lactamase activity increased to high levels in sputum from patients with CF during the course of piperacillin, ceftazidime, cefsulodin, or imipenem therapy. Aztreonam therapy lead to opposite results because the beta-lactamase activity decreased and aztreonam was able to mask beta-lactamase activity by acting as an inhibitor. All sputum beta-lactamases displayed characteristics indicative of a class I enzyme, identical to the beta-lactamases extracted from P. aeruginosa. The presence of beta-lactamase at such levels could lead to in vivo inactivation of beta-lactam antibiotics. This study supports the hypothesis that beta-lactamase production is an important in vivo resistance mechanism in P. aeruginosa-infected patients with CF.

Adolescent↗
PubMed1991

Evaluation of a small-scale method for the extraction of the K88 antigen from enterotoxigenic Escherichia coli.

A small-scale method for the extraction of the K88 major fimbrial subunit from enterotoxigenic Escherichia coli (ETEC) based on heat extraction has been developed. Variation in the buffer composition, time and temperature of extraction had negligible effect on the subsequent SDS-PAGE profile. There was, however, a correlation between the pH of the extraction buffer and the ensuing amount of K88 released. Reduction of the pH from 7 to 4 reduced by six-fold the amount of K88 released from the cells. We suggest that the relative stability of the K88 fimbriae at acid pH may influence the site of infection by ETEC in the intestine.

Antigens, Bacterial↗
PubMed1991

Structural studies on tazobactam.

Tazobactam (3, C10H12N4O5S) is an effective inhibitor of bacterial beta-lactamases. It crystallizes with unit cell dimensions a = 10.230 (2) A, b = 14.396 (2) A, and c = 17.291 (2) A in space group P2(1)2(1)2(1). Compared to the related inhibitor sulbactam (2), which lacks the triazole ring, crystalline tazobactam exhibits very similar beta-lactam geometry and the same S(1) envelope conformation of the thiazolidine ring. However, in both independent molecules of 3 a triazole ring nitrogen atom accepts an intermolecular hydrogen bond; similar interaction by this moiety of 3 with a hydrogen-bond donor on the enzyme, which is impossible for 2, could account for its enhanced inhibitory power. Semiempirical molecular orbital calculations show pronounced negative potential there. Molecular mechanics supports the hypothesis that the carboxyl group can rotate freely and the triazole ring can "flip".

Computer Simulation↗
PubMed1991

Effects of iron depletion and sub-inhibitory concentrations of antibodies on siderophore production by Staphylococcus aureus.

The effect of various sub-MICs of oxacillin and amikacin, alone or in combination, on siderophore production by Staphylococcus aureus growing under iron-depleted conditions was evaluated. Siderophore production varied greatly from strain to strain, ranging from 3.0 to 383.4 microM of Desferal equivalents. Siderophore production by some S. aureus strains was reduced significantly (P less than 0.001) under some sets of conditions. However, siderophore excretion in cultures with amikacin was sometimes equal to or greater than control cultures. Thus, at some sub-MICs, the bacteria tested were able to increase siderophore production under metabolic stress. This phenomenon may be a secondary effect of the antibiotics upon intermediary metabolism as a consequence of inhibition of peptidoglycan or protein synthesis. Alternatively the antibiotics may decrease the cellular association of siderophore molecules.

Amikacin↗
PubMed1991

Immunoblotting and ligand blotting of envelope structures of Staphylococcus aureus grown under iron insufficiency and in a sub-minimal inhibitory concentration of penicillin G.

Strip immunoblotting with specific, hyperimmune antisera and normal human sera (NHS), in conjunction with lectin and avidin ligand blotting and surface iodination, was used to investigate cell envelope components of phenotypes of Staphylococcus aureus NCTC 6571. Cocci were grown with a relatively slow doubling time (48 min) under iron sufficiency (Fe+) and iron insufficiency (Fe-), and with or without a sub-MIC of penicillin G (pen G), to approximate to in vivo conditions. Fe+ phenotypes demonstrated extra bands with prominent antigens at 48, 52 and 54 kD. Iron depletion or pen G simplified profiles, notably in the mid-range. In this particular S. aureus strain, protein A was ascribed a molecular mass of 35.5 kD and its detection was not affected by iron or pen G conditions. NHS reacted poorly with the Fe+ phenotype, supplying indirect evidence that this phenotype may not be common in vivo. Lectin blotting demonstrated the presence of glycosylated residues. Lectin affinities were not affected by the pen G treatment but in the Fe- phenotypes only, a 30 kD fucose-containing structure was seen. Avidin blotting visualised a major 86 kD binding site in all phenotypes which was not detected in immunoblotting. Whole cell radioiodination revealed that five major proteins of 21, 35.5, 48, 52 and 68 kD were surface-associated but their immunoreactivity depended upon the phenotype and source of sera.

Animals↗
PubMed1990

Characterisation of a siderophore from Acinetobacter calcoaceticus.

The Gram-negative bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant by 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.

Acinetobacter↗
PubMed1990

Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in-vivo selection of stable partially derepressed beta-lactamase producing strains.

The development of significant mechanisms of resistance to beta-lactam antibiotics in Pseudomonas aeruginosa in cystic fibrosis (CF) patients have been studied in ten CF patients during a two week course of anti-pseudomonal beta-lactam antibiotic therapy. Sputum samples were collected on days 1, 7 and 15. Entire homogenized sputum samples were examined directly for the number of bacteria resistant to different levels of antibiotics. This allowed the detection of pre-existing resistant subpopulations of bacteria as well as following the changes in beta-lactam antibiotic susceptibility during treatment. P. aeruginosa isolates were characterized by means of sero-grouping, phage- and pyocin-typing. Outer membrane proteins of paired sensitive and resistant strains were characterized. Sonicated extracts of cells were assayed for basal and induced beta-lactamase activity. Beta-lactamase activity was further characterized by isoelectric focusing and inhibition profiles. Our observations were in accordance with the hypothesis that the sensitive inducible population was overrun by the pre-existing resistant subpopulation, during treatment. The resistant in-vivo selected P. aeruginosa population exhibited stable partially derepression but the beta-lactamase inhibitor tazobactam restored beta-lactam antibiotic activity.

Adolescent↗
PubMed1990

Effect of in vivo growth conditions upon expression of surface protein antigens in Enterococcus faecalis.

Western blotting of whole-cell preparations of Enterococcus faecalis showed the protein-antigen profiles to be markedly influenced by growth conditions. The E. faecalis-specific antigens of 40 and 37 kDa, which are prominent in endocarditis, were strongly expressed following growth in serum or brain heart infusion, but not after growth in a chemically defined medium. The expression of these antigens in vivo was demonstrated in cells grown as a biofilm on silastic discs in the peritoneum of rabbits. These in vivo culture conditions may be useful in studying the pathogenesis of E. faecalis infections and the effectiveness of antibiotic therapy.

Animals↗
PubMed1990

Serological response in Enterococcus faecalis endocarditis determined by enzyme-linked immunosorbent assay.

Enterococcus (Streptococcus) faecalis expresses three species-specific surface protein antigens of molecular weights 73,000, 40,000, and 37,000. On Western blotting (immunoblotting), they were detected strongly by immunoglobulin G (IgG) in sera from patients with E. faecalis endocarditis, but not in sera from patients with other E. faecalis infections or with endocarditis due to other streptococci. We developed an enzyme-linked immunosorbent assay system to measure IgG, IgM, and IgA levels to these antigens and evaluated its potential as a serodiagnostic test for E. faecalis endocarditis. The test correctly diagnosed E. faecalis endocarditis in 15 of 16 cases. Of 10 cases of endocarditis due to other streptococci and 10 E. faecalis infections other than endocarditis, 9 and 8, respectively, gave negative results. The test should prove particularly useful in culture-negative cases, for which choice of appropriate antibiotic therapy for E. faecalis endocarditis is vital.

Antibodies, Bacterial↗
PubMed1990

Clomiphene treatment for women with unexplained infertility: placebo-controlled study of hormonal responses and conception rates.

One hundred and eighteen patients with unexplained infertility were treated with clomiphene (Clomid, Merrell) in a randomized placebo-controlled cross-over study lasting up to 3 months with each preparation. Compared with placebo, clomiphene led overall to a 66% increase in mid-luteal serum progesterone levels (from mean 43 nmol/l to 71 nmol/l, p less than 0.001) and a 53% rise in the 3-month cumulative conception rate (from 14.6% to 22.33%, p less than 0.05). The greatest relative increase in conception rates with clomiphene was in women with infertility lasting more than 3 years (3-month rates from 2.9% to 14.4%, p less than 0.05). Differences in conception rates were not related to the rises in progesterone, and there was no carryover effect of clomiphene into the following placebo cycles in terms of progesterone levels or conceptions. The findings suggest that couples with unexplained infertility of less than 3 years duration are essentially normal, but with more than 3 years duration there is often a subtle disorder of ovulation that is corrected by clomiphene therapy.

Adult↗
PubMed1989

Structural studies on bio-active compounds. Part XV. Structure-activity relationships for pyrimethamine and a series of diaminopyrimidine analogues versus bacterial dihydrofolate reductase.

The phenylpyrimidine derivative pyrimethamine and its congeners inhibit the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and are of interest as antiproliferative agents. In this study the equilibrium conformations of some pyrimethamine derivatives, and their interactions with Escherichia coli dihydrofolate reductase, were investigated using molecular modelling techniques. In each case the phenyl ring avoided coplanarity with the pyrimidine ring and attained a position approximately perpendicular to it, in agreement with crystal structures. A meta substituent could be placed either side of the pyrimidine plane, forming two non-equivalent, slowly interconverting solution conformations. Except for meta-azidopyrimethamine, both conformations of all the inhibitors were able to bind to the active site cleft of the enzyme with the diaminopyrimidine moiety, making the normal pattern of enzyme/inhibitor hydrogen bonds. One such conformation of the meta-azido compound failed to bind because of unacceptable steric clashes, whilst the other showed enhanced binding energy attributable to the occupation of a hydrophobic pocket by the azido group. The enhanced binding of 2,4-diamino-6-ethyl-5-phenylpyrimidine over its 6-methyl analogue was also related to attractive hydrophobic interactions.

Animals↗
PubMed1989

Iron depletion alters surface-associated properties of Staphylococcus aureus and its association to human neutrophils in chemiluminescence.

Staphylococcus aureus NCTC 6571 was grown in iron-depleted tryptone soya broth (Fe-TSB) to approximate to in vivo conditions, and in iron-rich TSB (Fe + TSB). Low iron effected a crucial decrease in surface hydrophobicity (SH) and a lack of supernatant Protein A (PrA). Iron availability did not affect PrA detection in immunoblotting and it was identified as a 35.5 kDa antigen in this strain. Fe-phenotypes lacked 34, 48 and 52 kDa antigens. In chemiluminescence, Fe-phenotypes appeared least vulnerable to phagocytosis despite opsonisation.

Antigens, Surface↗
PubMed1989

Binding of fibronectin and albumin to Enterococcus (Streptococcus) faecalis.

Binding of human fibronectin (FN) and albumin to Enterococcus (Streptococcus) faecalis was investigated. Scatchard analysis showed that the cells could bind a maximum of 1300 molecules of FN with an association constant of 4.8 x 10(6) M-1. Binding did not appear to involve lipoteichoic acid. Heating the cells or pretreatment with protease or periodate reduced the binding, suggesting the involvement of protein and/or carbohydrate-containing components as the surface receptor. The cells could bind a maximum of 50,000 molecules of albumin but with a much lower affinity than FN (association constant 10.8 x 10(3) M-1). Surface hydrophobicity of E. faecalis was markedly decreased by albumin but not by FN.

Albumins↗