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K L Powell

Publications and source records attributed to K L Powell.

At least 19 recordsLinked to original sources
PubMed1997

High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo[a]pyrene diol epoxide-DNA adducts.

Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle-regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site-containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA than for unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Interference of benzo[a]pyrene diol epoxide-deoxyguanosine adducts in a GC box with binding of the transcription factor Sp1.

Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site-specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high-affinity binding of Sp1 to non-Gc box sites.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Kinetics of the reaction of a potential chemopreventive agent, 2,6-dithiopurine, and its major metabolite, 2,6-dithiouric acid, with multiple classes of electrophilic toxicants.

Purinethiols are a class of potential cancer chemopreventive agents that exhibit nucleophilic scavenging activity against the carcinogenic electrophile benzo[a]pyrene diol epoxide (BPDE). Of the purinethiols tested previously, 2,6-dithiopurine (DTP), exhibited the highest scavenging activity for BPDE when tested either in vitro or in vivo. Sulfur-based nucleophiles are typically classified as "soft" nucleophiles, showing selectivity in nucleophilic substitution reactions for "soft", easily polarizable electrophiles. It was of interest to determine whether electrophilic toxicants other than BPDE react facilely with DTP, and whether 2,6-dithiouric acid (DUA), the major in vivo metabolite of DTP, also has scavenging activity. Four diverse toxicants tested in the present work, acrolein, melphalan, dimethyl sulfate, and cisplatin, all react facilely with DTP in vitro near neutral pH. These toxicants are expected to react as "soft" electrophiles. Furthermore, each of these compounds, as well as BPDE, reacts with DUA with rate constants comparable to the analogous rate constants for reaction with DTP. In contrast, several toxicants classified as "hard" electrophiles (ethyl methanesulfonate, methylnitrosourea, ethylnitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine) show no appreciable reaction with DTP. These results suggest that both DTP and its major metabolite act as "soft" nucleophiles in nucleophilic substitution reactions and may be effective in scavenging a wide range of toxicants that react as "soft" electrophiles.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Binding of the transcription factor, Sp1, to non-target sites in DNA modified by benzo[a]pyrene diol epoxide.

Covalent binding of the carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to DNA causes changes in the conformation of the DNA around the site of the adduct. However, the influence of such carcinogen-DNA adducts on interactions of the DNA with specific proteins has received little attention. Binding of the transcription factor, Sp1, to GC-box sequences in the promoter of the hamster adenosine phosphoribosyl transferase gene is a useful model system. Electrophoretic mobility shift assays, competition experiments and DNase I footprinting demonstrated specific binding of affinity-purified, human Sp1 to two adjacent GC-boxes in the promoter fragment. Unexpectedly, modification of this DNA fragment to high levels (approximately 7% of the nucleotides) with BPDE caused a substantial (5- to 10-fold) increase in the apparent affinity of Sp1. A heterologous DNA fragment that contained no GC-boxes did not compete for the binding of Sp1 to the promoter, unless it was previously modified with BPDE. In addition, two DNA fragments that contained no GC-boxes exhibited Sp1-dependent mobility shifts only when modified by BPDE. DNase I footprinting of the BPDE-modified, Sp1-bound promoter fragment did not reveal specific sites of binding, suggesting that numerous BPDE-DNA adduct sites can interact with the protein. A model in which Sp1 binding to non-target sites is enhanced by a static bend or an induced flexibility at the site of an adduct is discussed.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Toxicity and metabolism in mice of 2,6-dithiopurine, a potential chemopreventive agent.

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its facile reaction with the electrophilic ultimate carcinogen, benzo[a]pyrene diol epoxide, and other reactive electrophiles. Previous studies in mouse skin indicated almost complete inhibition of benzo[a]pyrene diol epoxide-induced tumorigenesis by DTP, suggesting the possible utility of this compound as a chemopreventive agent. However, little is known of the metabolism of DTP or of its possible long-term toxicity. Mice were fed diets containing up to 4% DTP in AIN-76A for a period of 7 weeks, and possible toxicity was monitored by weight gain and histopathological examination of all major tissues. No toxicity was observed at any dose of DTP. DTP was found to be a good substrate in vitro for two enzymes known to metabolize 6-mercapto-purine: xanthine oxidase and thiopurine methyltransferase. The in vitro metabolites were 2,6-dithiouric acid and an apparent monomethylated derivative, respectively. In vivo, the major urinary metabolite was 2,6-dithiouric acid, which attained levels as high as 34 mM in the urine of mice receiving the 4% DTP diet. DTP was also excreted unchanged in the feces and urine. DTP, 2,6-dithiouric acid, and an unidentified, relatively nonpolar metabolite were also detected in the serum of experimental animals. Although large interindividual variation in the serum DTP concentration was found, there was a dose-dependent increase in serum DTP as the dietary level of DTP was increased. These results suggest that neither toxicity nor metabolism will severely limit the utility of DTP as a chemopreventive agent.

Algorithms↗
PubMed1994

Human G-protein-coupled inwardly rectifying potassium channel (GIRK1) gene (KCNJ3): localization to chromosome 2 and identification of a simple tandem repeat polymorphism.

The gene encoding the human G-protein-coupled inwardly rectifying potassium channel designated GIRK1 (gene symbol, KCNJ3) was mapped to chromosome 2 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This assignment was confirmed by fluorescence in situ hybridization to metaphase chromosomes, and the gene was further localized to band 2q24.1. A highly informative simple tandem repeat DNA polymorphism of the form (CA)n was identified and used to localize KCNJ3 within the genetic map of the long arm of chromosome 2.

Animals↗
PubMed1994

Identification of a novel, N7-deoxyguanosine adduct as the major DNA adduct formed by a non-bay-region diol epoxide of benzo[a]pyrene with low mutagenic potential.

A metabolite of benzo[a]pyrene, 9-r,10-t-dihydroxy-7,8-c-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-III), that is not thought to be involved in carcinogenesis has nevertheless been shown to bind extensively to DNA in vitro. The adducts formed by this non-bay-region diol epoxide in Chinese hamster ovary cells are much less mutagenic than those formed by an isomeric diol epoxide that is carcinogenic. We have isolated and characterized three major adducts formed by in vitro reaction of BPDE-III with DNA. The major adduct, accounting for over half of the total is formed by reaction of BPDE-III with the N7 position of dGuo and is recovered after enzymatic digestion as an N7-Gua adduct. A second major adduct involves the N2 position of dGuo, while the third adduct is tentatively identified as a C8-substituted dGuo. Little or no reaction with deoxyadenosine residues is detected. The N7 adduct is unstable in DNA at 37 degrees C and is released as the modified base with a half-life of about 24 h. This adduct lability apparently leads to single-strand breaks and alkali-sensitive sites in the DNA and may account in part for some of the biological properties of BPDE-III adducts. This represents the first description of an N7-dGuo adduct that is formed in DNA as the major adduct by a diol epoxide derived from a carcinogenic polycyclic aromatic hydrocarbon.

Animals↗
PubMed1993

Effectiveness of buspirone on urine spraying and inappropriate urination in cats.

The most frequent type of behavior problem in cats for which veterinary consultation is sought is problem urination. Urine spraying and urine marking have been treated by use of long-acting progestins and diazepam, a benzodiazepine antianxiety drug. Effectiveness of the nonbenzodiazepine antianxiety drug, buspirone, in suppressing urine spraying and marking in 47 male and 15 female cats was evaluated. The effect of the drug in correcting inappropriate urination in 9 cats also was evaluated. Buspirone resulted in a favorable response (> 75% reduction) in 55% of cats treated for urine spraying or marking. There was no sex difference in effectiveness of the treatment, but cats from single-cat households responded favorably significantly (P < 0.001) less frequently than those from multiple-cat households. The 55% response rate was within the range of treatment effectiveness that has been reported for diazepam, and was greater than that reported for progestin. In contrast to diazepam, with which over 90% of treated cats resumed spraying or marking when the drug was gradually discontinued, only half of the cats treated with buspirone resumed spraying when the drug was discontinued after 2 months of treatment (P < 0.001). This difference between diazepam and buspirone in resumption of urine spraying was attributed to diazepam's induction of physiologic and behavioral dependency, not found with buspirone. Cats that resumed spraying were placed on long-term treatment ranging from 6 to 18 months. Buspirone also did not cause the adverse effects of sedation and ataxia, which commonly are seen with diazepam treatment. In cats treated for inappropriate urination, 56% returned to normal litter box usage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Review of research leading to new anti-herpesvirus agents in clinical development: valaciclovir hydrochloride (256U, the L-valyl ester of acyclovir) and 882C, a specific agent for varicella zoster virus.

Research leading to the new anti-herpesvirus compounds discussed here has come from three approaches. The first approach was directed towards improving the bioavailability of acyclovir by examining the potential of a variety of prodrugs, leading to the new compound valaciclovir hydrochloride. The second approach was to examine a large number of 5-substituted pyrimidines for activity against those viruses which were not as potently inhibited by acyclovir as are herpes simplex viruses, i.e., varicella zoster virus (VZV) and human cytomegalovirus (HCMV). This research led to the new chemical entity 882C for VZV. A third approach has been to examine drug combinations with acyclovir. This research led to the compound 348U, an inhibitor of herpes simplex virus ribonucleotide reductase which acts synergistically in combination with acyclovir. This manuscript will focus on the first two approaches leading to new compounds valaciclovir hydrochloride and 882C since Dr. Safrin details such background for 348U/acyclovir. Attempts to improve the bioavailability of acyclovir began a decade ago. Early prodrugs were compounds with alterations in the 6-substituent of the purine ring of acyclovir. The 6-amino congener required the cellular enzyme adenosine deaminase for conversion to acyclovir and the 6-deoxycongener was dependent on cellular xanthine oxidase for conversion. Neither of these prodrugs had a chronic toxicity profile in laboratory animals as good as acyclovir. Efforts were directed towards simpler esters and 18 amino acid esters were made. The pharmacokinetic profile of each prodrug was determined in rats by measuring the recovery of acyclovir in urine after oral dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyclovir↗
PubMed1993

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Amino Acid Sequence↗
PubMed1992

Association between the herpes simplex virus major DNA-binding protein and alkaline nuclease.

Herpes simplex virus encodes seven proteins which have been shown to be both necessary and sufficient for in vitro replication of origin-containing plasmids. We have shown previously that one of these proteins, the major DNA-binding protein mDBP, forms a complex with alkaline nuclease, which is not one of the seven essential proteins. In this study, we have employed immunological reagents and a series of deletion mutants to investigate this complex further. We have determined the regions of mDBP which are important in the formation of this complex, and we have shown that the intranuclear locations of alkaline nuclease and major DNA-binding protein overlap.

Animals↗
PubMed1992

Random mutagenesis of the thymidine kinase gene of varicella-zoster virus.

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260.

Amino Acid Sequence↗
PubMed1992

Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells.

Expression of the human cytomegalovirus (HCMV) (AD169) DNA polymerase gene under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus in Spodoptera frugiperda (Sf9) cells has provided a source of highly active CMV DNA polymerase. In extracts from CMV-infected cells, the CMV DNA polymerase is found strongly associated with an additional polypeptide, ICP36. This protein has been identified as the CMV homolog of the herpes simplex virus type 1 UL42 gene product and may have a similar function. We have expressed HCMV DNA polymerase and ICP36 in the same system and demonstrated that they interact to form a stable complex. Moreover, ICP36 functions to stimulate the DNA polymerase activity in a template-dependent manner. We have compared the activity of the recombinant DNA polymerase in the presence and absence of ICP36 on a number of DNA templates and measured the effect of the polymerase inhibitors phosphonoformic acid and acyclovir triphosphate.

Amino Acid Sequence↗
PubMed1991

Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction.

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.

Amino Acid Sequence↗
PubMed1991

High level expression of DNA polymerases from herpesviruses.

The DNA polymerase genes of human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) were inserted separately into the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV) by cotransfection of Spodoptera frugiperda (SF9) cells with baculovirus transfer vectors carrying the genes and AcNPV infectious DNA. Infection of SF9 cells with the recombinant viruses resulted in expression from the polyhedrin promoter of proteins of the expected Mrs. These proteins possessed DNA polymerase activities similar to that of the enzymes induced by the respective herpesvirus in infected cells, and were identified as HCMV and VZV DNA polymerase using inhibitors and specific antisera reactive with each enzyme.

Animals↗
PubMed1990

Antibacterial properties of saliva: role in maternal periparturient grooming and in licking wounds.

Canine saliva was tested for its bactericidal effects against pathogens relevant to the presumed hygienic functions of maternal grooming of the mammary and anogenital areas and licking of wounds. Both female and male saliva were bactericidal against Escherichia coli and Streptococcus canis but only slightly, and nonsignificantly, bactericidal against coagulase positive staphylococcus and Pseudomonas aeruginosa. E. coli is the cause of highly fatal coliform enteritis of neonatal mammals and E. coli and S. canis are the main pathogens implicated in neonatal septicemia of dogs. The bactericidal effects of saliva would facilitate the hygienic function of maternal licking of the mammary and anogenital areas in protecting newborns from these diseases. E. coli and S. canis along with coagulase positive staphylococcus and P. aeruginosa are among the common wound contaminants of dogs. Wound licking, and the application of saliva, would thus reduce wound contamination by E. coli and S. canis. The resistance of staphylococcus to bactericidal effects of saliva may be a factor in the high frequency (46 percent) with which coagulase positive staphylococcus was isolated from wounds compared with much lower frequency (9-17 percent) with which E. coli and S. canis were isolated.

Animals↗