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Biomedical subjects

J A Fyfe

Publications and source records attributed to J A Fyfe.

At least 55 records · Page 3Linked to original sources
PubMed1984

Chromosomal loci associated with antibiotic hypersensitivity in pulmonary isolates of Pseudomonas aeruginosa.

492a and 492c were two strains of Pseudomonas aeruginosa isolated from the sputum of a patient with cystic fibrosis. The strains were closely related but expressed different antibiograms. 492c was hypersensitive (10-100 times more sensitive than 492a) to the beta-lactam antibiotics carbenicillin, methicillin, flucloxacillin, mecillinam and cefuroxime and the non-beta-lactam, nalidixic acid. 492c also showed enhanced sensitivity (4-8 times more sensitive than 492a) to chloramphenicol, trimethoprim and novobiocin. 492a and PAO8 expressed similar levels of antibiotic resistance, except for trimethoprim, to which 492a was five times more sensitive than PAO8. Two genes associated with antibiotic hypersensitivity were mapped in the 30 min region of the chromosome, by means of R68.45-mediated plate matings between a Leu - mutant of 492c and PAO8, followed by transductional analysis using phage F116L. The first of these genes, blsA1, was closely linked to nalB, and in a PAO background, was associated with hypersensitivity to the beta-lactams and a moderate increase in sensitivity to chloramphenicol, trimethoprim, nalidixic acid and novobiocin. A further increase in sensitivity to the latter three antibiotics was associated with the second gene, tpsA1, which mapped between ser-3 and hisV. This gene could also be transferred to PAO from 492a, thus 492c could have arisen from 492a in vivo following a single chromosomal mutation at the blsA locus. Isolation of a blsA mutant of PAO969 provided further evidence for this theory.

Anti-Bacterial Agents↗
PubMed1984

Revised pyocin typing method for Pseudomonas aeruginosa.

In the Gillies and Govan method of pyocin typing for Pseudomonas aeruginosa a cross-streaking technique was used, and 105 main types and 25 subtypes were identified by the patterns of inhibition observed on 13 indicator strains. Disadvantages of the technique included the need to remove test strain growth before application of the indicator strains, the 48-h period needed to obtain a result, and the inability to reliably type mucoid P. aeruginosa. Recent studies have enabled us to overcome these disadvantages and significantly improve the speed and application of pyocin typing. Our revised technique utilizes the same 13 indicator strains which are already used internationally. Test strains were rapidly applied to the surface of agar plates with a multiple inoculator. After incubation for 6 h and exposure to chloroform, the indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, the pyocin type was recognized by inhibition of particular indicator strains. Additionally, the activity of particulate (R and F) and nonparticulate (S) pyocins could be distinguished on the basis of inhibition zone size, which thus allowed further discrimination. The revised technique allows typing within 24 h, increases the number of identifiable types, and can be used to type mucoid strains.

Bacteriocins↗
PubMed1983

Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine.

Virus-coded thymidine (dThd) kinases were purified by affinity chromatography from a parental strain (SC16) and two strains (SC16 B3 and SC16 S1) of herpes simplex virus, Type 1, with altered drug sensitivities. These latter two strains were less sensitive, respectively, to E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and to both BrVdUrd and 9-(2-hydroxyethoxymethyl)guanine (acyclovir). The enzymes were characterized with respect to physical and catalytic properties. The enzyme from SC16 B3 was very similar to the parental enzyme except in its substrate specificity and kinetic constants. It catalyzed the phosphorylation of BrVdUrd at a relative rate that was 110% of the rate with dThd versus a relative rate of 140% with the parental enzyme. The apparent Km value for BrVdUrd was 6 microM versus 0.1 microM for the parental enzyme. The reaction kinetics with acyclovir were similar for the two enzymes. The SC16 B3 enzyme catalyzed the phosphorylation of dTMP, but at only 2% the efficiency of the parental enzyme; phosphorylation of the monophosphate of BrVdUrd (BrVdUMP) was not detected with the SC16 B3 enzyme. The enzyme from the SC16 S1 variant had a much narrower phosphate acceptor specificity than the enzyme from the parental virus. BrVdUrd was a substrate but with a relative rate of 30% and an apparent Km value of 4 microM; acyclovir was neither detectably phosphorylated nor a good inhibitor. BrVdUMP was not detectably phosphorylated. The relative efficiencies of the two variant enzymes for acyclovir phosphorylation correlated well with the sensitivities of the viruses to this compound. In contrast, the relative efficiencies of the second phosphorylation step (BrVdUMP to BrVdUDP) were most consistent with the sensitivities of the viruses to BrVdUrd.

Acyclovir↗
PubMed1982

Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus.

Acyclovir inhibited the replication of a varicella-like simian virus (DHV-1) in cell culture (Vero cells) with an ED50 of 38 +/- 2 microM. The activation of acyclovir in this cell culture system was compared with that in the cell system with human varicella zoster virus (VZV). Extracts of cells infected with DHV-1 catalyzed the phosphorylation of acyclovir. The phosphorylation was inhibited by dThd, suggesting the catalyst was a dThd kinase. Electrophoresis of cytosol fractions on polyacrylamide gels corroborated the existence of a virus-associated dThd kinase. This enzyme copurified with an acyclovir-phosphorylating activity. The enzyme catalyzed the phosphorylation of acyclovir at a greater relative rate than that with the VZV enzyme, but with a higher apparent Km value for acyclovir. The relative efficiencies for the two enzymes with acyclovir were similar. Anabolic studies with cells infected with DHV-1 and incubated with [14C]acyclovir indicated that triphosphate of acyclovir did accumulate. The results indicate that acyclovir is activated in cells infected with DHV-1 in a manner similar to that in cells infected with VZV.

Acyclovir↗
PubMed1982

Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus.

A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 microM acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay. The capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovir-sensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumable due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.

Acyclovir↗
PubMed1982

Differential phosphorylation of (E)-5-(2-bromovinyl)-2'-deoxyuridine monophosphate by thymidylate kinases from herpes simplex viruses types 1 and 2 and varicella zoster virus.

5-(2-Bromovinyl)-2'-deoxyuridine (BrVdUrd) is a potent and selective inhibitor of herpes simplex virus Type I (HS-I) and varicella zoster (VZ) virus replication but is much less potent against herpes simplex virus Type II (HS-II) replication. A possible enzymatic basis for this difference is reported here to involve the virus-coded dThd-dTMP kinases (EC 2.7.1.75) from the three virus strains. The thymidine kinases from the three virus strains were purified by affinity chromatography. In addition to catalyzing the phosphorylation of nucleosides, each of the three purified enzymes catalyzed the phosphorylation of thymidylate to its diphosphate but at strikingly different rates. The relative amounts of extractable virus-coded thymidylate kinases were estimated to be 100/2/40 for cells infected with HS-I, HS-II, and VZ viruses, respectively. Extracts of cells infected with HS-I virus catalyzed the phosphorylation of the monophosphate of BrVdUrd to its diphosphate. In contrast, the product was not detected with extracts from cells infected with HS-II virus. THe ratios of rates with 0.5 mM BrVdUrd monophosphate versus 0.1 mM dTMP as substrates for each of the purified dThd-dTMP kinases from HS-I, HS-II, and VZ viruses and the dTMP kinase from host cells were, respectively, 0.09, less than 0.002, 0.03, and less than 0.0002. These observations correlate with the relative sensitivities of these viruses to BrVdUrd in cell culture and suggest that, if BrVdUrd exerts its effect as a triphosphate, the inefficient phosphorylation of the monophosphate contributes to the insensitivity of HS-II virus to this agent.

Bromodeoxyuridine↗
PubMed1981

Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina.

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.

Alginates↗
PubMed1981

Heterogeneity of antibiotic resistance in mucoid isolates of Pseudomonas aeruginosa obtained from cystic fibrosis patients: role of outer membrane proteins.

Mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients are very heterogeneous and include a class which is hypersusceptible to carbenicillin (minimum inhibitory concentration, less than or equal to 1 microgram/ml). Hypersusceptible mucoid P. aeruginosa isolates were found in 12 of 22 cystic fibrosis patients examined. In cystic fibrosis patients having both resistant and hypersusceptible mucoid strains, 24 of 54 mucoid colonies obtained from a sputum sample were found to belong to the hypersusceptible class. In most instances, hypersusceptible and resistant strains isolated from the same sputum sample were indistinguishable, aside from their antibiotic susceptibilities, by classical methods. A particular pair of mucoid isolates (one hypersusceptible and one resistant) was chosen for further study. The hypersusceptibility was not limited to carbenicillin but was found to extend to other penicillins, tetracycline, and trimethoprim but not to the aminoglycosides gentamicin and tobramycin. The hypersusceptibility of the mucoid strain was found to be unrelated to amount or ability to synthesize alginate. The hypersusceptible strain was found to have two additional outer membrane proteins (32,000 and 25,000 daltons) as compared with the resistant strain. The 32,000-dalton protein, termed protein N1, was found to be correlated to the hypersusceptibility phenotype, as all spontaneous mutants of the hypersusceptible mucoid strain which were capable of growing in the presence of 50 microgram of carbenicillin per ml had lost the 32,000-dalton outer membrane protein. The possible origins of the hypersusceptibility phenotype and the implications of the heterogeneity of mucoid P. aeruginosa in the pathogenesis of P. aeruginosa are discussed.

Anti-Bacterial Agents↗
PubMed1980

Alginate synthesis in mucoid Pseudomonas aeruginosa: a chromosomal locus involved in control.

Mucoid variants of Pseudomonas aeruginosa isolated in vitro or in vivo could be classified into two phenotypic groups based on whether alginate was produced on a chemically defined medium. Mucoid strains yielded lowe recombination frequencies than the non-mucoid parent when used as donors in FP2-mediated plate matings. The mucoid characteristic (muc) was co-inherited by a proportion of recombinants selected for the inheritance of chromosomal markers his-5075+ or cys-5605+. The results of further experiments using either a mucoid recipient or a mucoid donor carrying plasmid R68.45 suggested that the control of alginate production in P. aeruginosa involves at least one chromosomal locus.

Alginates↗
PubMed1979

Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.

The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.

Animals↗
PubMed1977

Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine.

A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).

Antiviral Agents↗