Nucleotide sequence and expression in Escherichia coli of the recA gene of Neisseria gonorrhoeae.

The nucleotide sequence of the recA gene of Neisseria gonorrhoeae MS11 has been determined. The product of this gene can act as a recombinase in Escherichia coli, but does so with a decreased efficiency, probably because of the formation of mixed multimers with the equivalent E. coli protein.

Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide.

Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers.

The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide.

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP).

Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa.

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P.

Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir.

To identify the nucleotide changes that occur in drug-induced thymidine kinase (TK) mutants of herpes simplex virus type 2 (HSV-2), we compared the nucleotide sequences of the tk genes of two mutant HSV-2 clones isolated from a patient who had been treated with acyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV] with the nucleotide sequence of the parental TK+ HSV-2(8703) strain isolated from the same patient. One of the mutants, TK-altered (TKA) HSV-2(9637), was ACV resistant but induced the incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. The nucleotide sequence of the tk gene of mutant TKA HSV-2(9637) had a single change (G to A) at nucleotide 668, which would cause an arginine-to-histidine substitution at amino acid residue 223 of the TK polypeptide.

3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus.

We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread.

Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity.

In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.

Ribavirin antagonizes the effect of azidothymidine on HIV replication.

Azidothymidine and ribavirin both inhibit replication of human immunodeficiency virus in vitro and show promise of clinical utility in patients infected with this virus. In this study, the possible interactions of these drugs were examined in vitro, and a reproducible antagonism between azidothymidine and ribavirin was found to occur under a variety of experimental conditions. The mechanism responsible for this antagonism appeared to be inhibition of azidothymidine phosphorylation by ribavirin.

Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U).

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or most fungal pathogens.

The Myers-Briggs type indicator and coronary heart disease.

Researchers have for many years attempted to establish a relationship between coronary heart disease (CHD) and personality type. In our study, 103 subjects completed Form G of the Myers-Briggs Type Indicator (MBTI). Comparisons were made between 93 CHD patients and an age-appropriate control group (Group C) on each of the four MBTI dimensions: Extraversion-Introversion, Sensing-Intuition, Thinking-Feeling, and Judging-Perceiving.

A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.

We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed.

Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J.

Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.

A group of compounds was prepared in which variations of the ring portion of the acyclovir (ACV) structure were made. These modifications included monocyclic (isocytosine, triazole, imidazole), bicyclic (8-azapurine, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine) and tricyclic (linear benzoguanine) congeners. The derivatives were evaluated against herpes simplex virus type 1 (HSV-1) by the plaque-inhibition and plaque-reduction methods with only the 8-azapurine analogue 28 showing some activity.

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation.

A cytoplasmic 5'-nucleotidase (EC 3.1.3.

Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus.

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection.

Orthophosphate determination in the presence of high concentrations of ATP.

A method to measure orthophosphate which contaminates samples of ATP was developed. Concentrations of orthophosphate as low as 0.4% of the ATP concentration were determined using a zinc-molybdate reagent [D.

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.

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.

Heterogeneity and reduction in pulmonary clearance of mucoid Pseudomonas aeruginosa.

Mucoid, alginate-producing Pseudomonas aeruginosa isolated from patients with cystic fibrosis were studied to determine the extent of heterogeneity of the isolates within individual sputa. Considerable heterogeneity involving cultural requirements for mucoid colonial growth was observed, and sensitivity to the beta-lactam antibiotic carbenicillin was also variable. For determining if the presence of alginate increased pulmonary survival of the bacteria, groups of rats were infected by transtracheal instillations of equivalent numbers of mucoid or nonmucoid P.

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.

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.