Biomedical subjects

G E Wright

Publications and source records attributed to G E Wright.

At least 55 records · Page 3Linked to original sources

PubMed1994

Inhibitor analysis of calf thymus DNA polymerases alpha, delta and epsilon.

Quantitative effects of inhibitors of the replicative DNA polymerases (pol) alpha, delta and epsilon from calf thymus are reported under similar assay conditions. Carbonyldiphosphonate was a competitive inhibitor of pols delta and epsilon, with 4- to 6-fold selectivity compared to pol alpha. Aphidicolin inhibited pols alpha and delta with 6- to 10-fold selectivity compared to pol epsilon. The 'butylphenyl' nucleotides, BuPdGTP and BuAdATP, inhibited pol alpha with at least 1000-fold selectivity compared to pols delta and epsilon. The use of these inhibitors under similar assay conditions permits the discrimination of the three enzymes.

Animals↗

PubMed1994

Identification, partial purification and inhibition by guanine analogues of a novel enzymic activity which phosphorylates guanosine to GMP in the protozoan parasite Eimeria tenella.

From oocysts of the protozoan parasite Eimeria tenella, responsible for avian coccidiosis, we have partially purified and characterized a novel enzymic activity which specifically phosphorylates guanosine to GMP. The enzyme is able to use several phosphate donors, in the order: acetyl phosphate (Ac-P) > ATP > UTP > CTP > phosphoribosyl pyrophosphate (PRPP) > dUTP > or = dATP. The low specificity of this enzyme for the phosphate donor suggested that it be named guanosine phosphotransferase (GPTase). This enzyme is biochemically distinct from the previously described adenosine kinase (AK) and hypoxanthine/xanthine/guanine phosphoribosyltransferase (HXGPRTase), and may enable the parasite to synthesize guanine nucleotides under conditions of imbalance between adenine and guanine nucleotides. Because of its possible role in the purine salvage pathways, we have studied the effect of several guanine and guanosine analogues, recently synthesized in our laboratory, on the activity of GPTase in vitro. GPTase is specifically inhibited in the micromolar range by several substituted N2-phenylguanine bases. These results indicate that, as previously found for AK and HXGPRTase, GPTase could be a potential target for antiparasitic chemotherapy.

Adenosine Kinase↗

PubMed1994

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.

Adenine Nucleotides↗

PubMed1993

Herpes simplex virus type 1 uracil-DNA glycosylase: isolation and selective inhibition by novel uracil derivatives.

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

Cell Nucleus↗

PubMed1992

Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide.

An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods. Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry. After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination. The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer. E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer. E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.

Base Sequence↗

PubMed1991

The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization.

Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex. Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini. In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.

Adenosine Triphosphate↗

PubMed1991

Hospital choice of Medicare beneficiaries in a rural market: why not the closest?

As part of a larger study of hospital choice, the travel patterns of more than 12,000 Medicare beneficiaries residing in three overlapping rural areas were examined. During 1986 these Medicare beneficiaries were admitted to one of 53 hospitals in an area that encompassed parts of Minnesota, North Dakota, and South Dakota. Information on ZIP code of residence, closest hospital, and hospital of admission were used to analyze hospital choices of the Medicare rural elderly residing in this area. To summarize their travel patterns, the admitting hospital was categorized based on whether it was urban or rural, its size and whether or not it was the closest facility. Findings indicated that 60 percent of these rural Medicare beneficiaries used hospital services at their closest rural hospital, regardless of its size. However, 79 percent of those whose closest hospital was larger than 75 beds used it, while only 54 percent of those whose closest rural hospital was fewer than 75 beds obtained services there. Overall, 30 percent of those residing in this rural market area went to an urban hospital. These patterns appeared to reflect an evaluation by the physician and/or individual of the relative attractiveness of the local hospital versus alternatives available, as well as the individual's characteristics. Travel patterns varied by the beneficiary's age as well as his or her relative complexity of illness, as measured by a Disease Staging methodology. Findings have implications for the provision and financing of hospital services in rural areas.

Aged↗

PubMed1991

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii.

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO(2)). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.

Journal Article↗

PubMed1991

Medicaid support of alcohol, drug abuse, and mental health services.

Medicaid expenditures for alcohol, drug abuse, and mental health (ADM) services in 1984 were examined for the States of California and Michigan. Persons receiving such services constituted 9 to 10 percent of the total Medicaid population in the two States and accounted for 22 to 23 percent of total Medicaid expenditures. ADM expenditures were 11 to 12 percent of the total. Although the two States had similar proportions of overall expenditures for these services, Michigan appeared to emphasize inpatient psychiatric care, while California emphasized ambulatory and nursing home care. Based on the experience of the two States, national Medicaid expenditures for ADM services exclusive of long-term care were estimated to be $3.5 to $4.9 billion in 1984, two to three times the level suggested by earlier estimates.

Adult↗

PubMed1991

Predicting hospital choice for rural Medicare beneficiaries: the role of severity of illness.

Previous research has confirmed that desirable hospital attributes as well as increased distance, or travel time, have an impact on hospital choice. These studies have become increasingly sophisticated in modeling choice. This study adds to the existing literature by estimating the effect of both hospital and individual characteristics on hospital choice, using McFadden's conditional logit model. Some patient characteristics have not previously been accounted for in this type of analysis. In particular, the effect of a patient's complexity of illness (as measured by Disease Staging) on the choice of hospital is taken into account. The data consist of over 12,000 Medicare discharges in three overlapping rural market areas during 1986. The hospital choice set was aggregated into seven groups of urban and rural hospitals. Results indicate that rural Medicare beneficiaries tend to choose hospitals with a large scope of service and with teaching activity over those with a lower scope of service and no teaching activity, holding other factors constant. Distance is a deterrent to hospital choice, especially for older Medicare beneficiaries. The more complex cases tend to choose larger urban and rural hospitals over small rural hospitals more often than less complex cases do.

Age Factors↗

PubMed1991

Sterility testing of antimicrobial-containing injectable solutions prepared in the pharmacy.

The need for sterility testing of antimicrobial-containing injectable solutions is discussed and specific testing methods are described. Despite their antimicrobial activity, antimicrobial-containing injectable drug products are not necessarily self-sterilizing and can become contaminated. In addition to practicing aseptic technique, pharmacists must perform end-product sterility testing on intravenous solutions to ensure their sterility. The United States Pharmacopeia provides guidelines for the performance and validation of two sterility test methods: membrane filtration and direct transfer to culture media. Membrane filtration is the method of choice for sterility testing of many antimicrobial-containing injectable solutions. After the test article is filtered, the membrane is rinsed with sterile fluid to remove residual antimicrobial agent, cut into two portions, and immersed in two types of culture medium. Visible turbidity of a sample within the appropriate incubation period indicates the presence of a contaminating microorganism. Closed filtration systems minimize false-positive results. In the direct transfer method, samples of the test article are directly inoculated into vessels of culture media, and antimicrobial activity is eliminated by dilution or by deactivation with chemical or enzymatic agents. Sterility testing as well as aseptic technique is needed to ensure the sterility of antimicrobial-containing injectable solutions.

Anti-Infective Agents↗

PubMed1990

Development of novel inhibitor probes of DNA polymerase III based on dGTP analogs of the HPUra type: base, nucleoside and nucleotide derivatives of N2-(3,4-dichlorobenzyl)guanine.

6-(p-Hydroxyphenylhydrazino)uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific class III DNA polymerase (pol III) of Gr+ bacteria. Although formally a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. We describe the successful conversion of the H2-HPUra inhibitor prototype to a bona fide purine, using N2-(benzyl)guanine (BG) as the basis. Structure-activity relationships of BGs carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl)guanine (DCBG) as a nucleus equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of B. subtilis DNA pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization.

Base Sequence↗

PubMed1990

(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins.

5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5'-[beta,gamma-(Difluoromethylene)triphosphates], including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate. The 5'-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-[(alpha,beta-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphospate]. 2'-Deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.

Bacillus subtilis↗

PubMed1990

The roles of DNA polymerases alpha and delta in DNA replication.

The identities and precise roles of the DNA polymerase(s) involved in mammalian cell DNA replication are uncertain. Circumstantial evidence suggests that DNA polymerase alpha and at least one form of DNA polymerase delta, that which is stimulated by Proliferating Cell Nuclear Antigen, catalyze mammalian cell replicative DNA synthesis. Further, the in vitro properties of polymerases alpha and delta suggest a model for their coordinate action at the replication fork. The present paper summarizes the current status of DNA polymerases alpha and delta in DNA replication, and describes newly available approaches to the study of those enzymes.

Animals↗

PubMed1990

Deoxyribonucleotide analogs as inhibitors and substrates of DNA polymerases.

Inhibitory and substrate properties of analogs of deoxyribonucleoside triphosphates toward DNA polymerases are reviewed. A general introduction is followed by a description of DNA polymerases and the reaction that they catalyze, and sites at which substrate analogs may inhibit them. Effects of modifications in the major family of compounds, nucleotide derivatives, at the base, sugar and triphosphate portions of the molecule, are summarized with respect to retention of substrate properties and generation of inhibitory properties. Structure-activity relationships and the basis of selectivity in the second family of compounds, deoxyribonucleotide mimics, are also presented. Conclusions are drawn regarding the structural basis of inhibitor selectivity and mechanism, relationship between in vitro and in vivo effects of inhibitors, and the promise of inhibitors as probes for study of active sites of DNA polymerases.

Animals↗

PubMed1990

Specific inhibitors of herpes simplex virus thymidine kinase diminish reactivation of latent virus from explanted murine ganglia.

Two specific inhibitors of herpes simplex virus thymidine kinase, N2-phenyl-2'-deoxyguanosine and N2-(m-trifluoromethylphenyl)guanine, were tested for their ability to inhibit the reactivation of virus from explant cultures of latently infected murine trigeminal ganglia. Both compounds significantly diminished the frequency of reactivation compared with that of untreated controls.

Animals↗

PubMed1989

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta.

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.

Animals↗

PubMed1989

A kinetic study of rat recombinant DNA polymerase beta: detection of a slow (hysteretic) transition in polymerase activity and inhibition by butylphenyl-deoxyguanosine triphosphate.

We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase beta with synthetic polynucleotides. Nucleotide incorporation is biphasic; an initial burst of activity decays exponentially to a lower steady-state velocity. This slow transition in polymerase activity is not due to substrate depletion, abortive complex formation, or enzyme inactivation. The data are consistent with description of the beta-polymerase as a hysteretic enzyme, a finding which provides a potential explanation for the non-hyperbolic kinetics which have been reported previously for this polymerase. We have also found, in contrast to some previous data, that the nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPdGTP), is an inhibitor of the beta-polymerase. When poly(dC).oligo(dG) is used as template.primer, inhibition of the initial velocity is competitive with dGTP with a Ki of 1.25 microM. On activated DNA, however, beta-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase delta; 100 microM BuPdGTP is required to inhibit the initial velocity of a dGTP-deficient, truncated assay. Finally, we demonstrate that, in addition to its inhibition of initial velocity, BuPdGTP also modulates both the rate constant of the slow transition in polymerase activity, and the steady-state velocity of the beta-polymerase.

Animals↗