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Biomedical subjects

C M King

Publications and source records attributed to C M King.

At least 91 records · Page 5Linked to original sources
PubMed1991

Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide.

We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.

Base Sequence↗
PubMed1990

Skin problems associated with routine wearing of protective gloves in dental practice.

Guidelines on cross-infection control recommend the wearing of operating gloves by dental practitioners whilst carrying out routine examinations and treatment on all patients. However, there are a number of dermatological problems that may be associated with the routine wearing of protective gloves in dental practice. This paper describes the aetiology and clinical features of three types of contact dermatitis, ie irritant contact dermatitis, allergic contact dermatitis and contact urticaria. The management of these skin conditions is discussed. Advice is given concerning routine handcare for all dental practitioners.

Dentists↗
PubMed1990

Mutagenesis by site-specific arylamine adducts in plasmid DNA: enhancing replication of the adducted strand alters mutation frequency.

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗
PubMed1990

Comparative survival of aminobiphenyl- and aminofluorene-substituted plasmid DNA in Escherichia coli Uvr endonuclease deficient strains.

pBR322 plasmid DNA, randomly substituted with arylamine moieties, was introduced into Escherichia coli Uvr endonuclease deficient strains. Plasmid survival was determined by selection in the presence of ampicillin. Modification of plasmid DNA with N-acetoxy-N-trifluoroacetylaminobiphenyl yielded primarily N-(deoxyguanosin-8-yl)-4-aminobiphenyl residues. Reaction of DNA with N-acetoxy-N-acetylaminobiphenyl produced only N-(deoxyguanosin-8-yl)-4-acetylaminobiphenyl adducts. The aminobiphenyl (ABP) and acetylaminobiphenyl adducts reduced the ability of the plasmid DNA to transform E. coli to approximately the same extent in a wild-type strain. In uvrA, uvrB and uvrC, i.e. Uvr endonuclease deficient strains, both adducts produced equivalent decreases in survival, however, the reduction in survival was much more pronounced in the uvr- cells than in the wild-type strain. A similar pattern of toxicity was observed with plasmids carrying N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts, although the acetylaminofluorene adduct was approximately 5-fold more effective in reducing the biological activity of the plasmid. In contrast, the deacetylated aminofluorene (AF) lesion, N-(deoxyguanosin-8-yl-2-aminofluorene, exhibited relatively little effect on plasmid survival in uvrA and uvrB cells as compared to the wild-type strain, even though the survival of both ABP and AF adducts was essentially similar in the uvrC and wild-type strains. These data demonstrate that (i) both the deacetylated and acetylated lesions are subject to repair by the Uvr endonuclease complex, and (ii) the presence of the N-acetyl group is not the sole determinant of the differential effects of arylamine adducts in uvr cells. These observations provide indirect evidence that both the N-acetyl and aryl moieties of these adducts alter the conformation of DNA.

Acetylation↗
PubMed1990

A case of actinic prurigo and solar urticaria.

Actinic prurigo and solar urticaria are uncommon chronic idiopathic photodermatoses in the United Kingdom. To our knowledge, their occurrence in the same patient has not hitherto been described.

Child↗
PubMed1990

Orofacial granulomatosis associated with delayed hypersensitivity to cobalt.

Orofacial granulomatosis is a distinct clinical and pathological entity characterized by swelling of the lips and lower half of the face. Ulceration of the oral mucosa may also occur. Granulomas are seen histologically. Orofacial granulomatosis may occur in the Melkersson-Rosenthal syndrome, granulomatous cheilitis of Miescher, oral Crohn's disease, sarcoidosis and focal dental sepsis. The increased prevalence of atopy in patients with orofacial granulomatosis and the association with food intolerance suggests the possibility of a role for allergy in at least some cases.

Child↗
PubMed1990

Continuity of care in a family practice residency program. Impact on physician satisfaction.

One of the primary benefits of continuity of care is its influence upon patient and physician satisfaction. This prospective pilot study involved a cohort of 14 second- and third-year family practice residents and 4 full-time faculty at a community hospital-based family practice residency in Cleveland, Ohio. Rates of continuity that physicians experience were calculated using the usual provider continuity (UPC) measure of continuity, and were correlated with physician satisfaction with outpatient care using a practice satisfaction scale (PSS) developed specifically for this purpose. Residents and faculty were also asked to rank order the importance of several aspects of outpatient care, including continuity of care. The results indicate that both residents and faculty value continuity of care highly compared with other aspects of outpatient care. The average continuity rates were 59% for second-year residents, 54% for third-year residents, and 82% for faculty. The UPC continuity measure correlated highly with the PSS scores (corrected r2 = .55; P less than .001). The data support the hypothesis that continuity of care with patients is an important determinant of resident and faculty physician satisfaction with their outpatient experience.

Adolescent↗
PubMed1989

Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites.

Two related carcinogen adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) or N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into the lacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA. The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct. These site-specifically modified templates were transfected into competent wild-type Escherichia coli cells (JM103) and a uvrA strain (SMH12). The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective beta-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region. DNA sequencing was used to characterize 179 of the mutants obtained. We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct. A specific frameshift (+1G) was also observed at a displaced site. All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide. Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts. The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression.

2-Acetylaminofluorene↗
PubMed1989

Mutations induced by aminofluorene-DNA adducts during replication in human cells.

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we treated a shuttle vector, pZ189, carrying the supF gene as the target for mutations with N-acetoxy-N-trifluoroacetyl-2-aminofluorene (N-AcO-TFA-AF). The plasmids were allowed to replicate in human cell line 293, and the progeny plasmids were examined for the frequency and kinds of mutations induced in supF, as well as their specific location in the sequence of the supF gene. The plasmids were reacted with N-AcO-TFA-AF so as to obtain the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), the principal adduct formed in DNA when mammalian cells are exposed to reactive derivatives of 2-acetylaminofluorene (AAF), including N-acetoxy-2-acetylaminofluorene. The results showed there was a linear relationship between the number of dG-C8-AF adducts per plasmid and the frequency of supF mutants induced. DNA sequencing of 47 independent mutants obtained from doses of N-AcO-TFA-AF that increased the frequency of mutants 9-15 times the background frequency and three independent mutants from lower doses showed that 92% contained point mutations, i.e. changes affecting one, or two, or three nearby bases, and that all of these point mutations involved G.C base pairs. Ninety eight percent of the point mutations were base substitutions, predominantly G.C----T.A transversions. 46% of these mutations occurred at four out of the 85 bp in the target gene (hot spots). The most prominent mutation hot spot was also the most prominent hot spot for adduct formation as judged by the frequency of termination of in vitro polymerization by the Klenow fragment on N-AcO-TFA-AF-treated plasmids.

2-Acetylaminofluorene↗
PubMed1989

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder.

N-Acetoxyarylamines are reactive metabolites that are implicated in the initiation of the carcinogenic process by some N-substituted aryl compounds. The objective of this study was to explore the relationship between the production of these reactive species and N-acetylation (NAT), a reaction previously demonstrated to be polymorphic in the human. Human liver and urinary bladder mucosa samples were frozen within 4-8 h post mortem. These tissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii) intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF. Cytosolic AHAT and OAT showed partial inhibition by paraoxon. The ratio of paraoxon insensitive AHAT to OAT to NAT of PABA to NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosols from frozen livers. Freezing of the cytosol resulted in extensive loss of activities. All four of these cytosolic enzyme activities exhibited a similar polymorphic response. Microsomal deacetylation showed a monomorphic response. Similar to the liver, urinary bladder epithelial cells also catalyzed the same reactions. However, the OAT and AHAT activities were detected mainly in microsomes. These data suggest that phenotypically rapid acetylators have a greater biochemical potential for the metabolic activation of aromatic amines by pathways that involve O-acetylation.

Acetylation↗
PubMed1989

Delayed patch test reactions at days 7 and 9.

403 consecutive patients were examined on days 2, 4, 7, and 9 following application of patch tests. Late positive reactions, i.e., those which manifested after day 4, were seen in 29 patients (7.2%). The allergens which most commonly produced late positive reactions were neomycin (7), potassium dichromate (4), and cobalt (3). Paired patch test readings on days 4 and 7 were more reliable than those on days 2 and 4. If only 1 patch test reading were performed, a single reading on day 4 would have been most useful. The reading on day 9 proved to be of limited value. Of 11 patients who were retested after an interval of more than 6 months, 7 showed earlier eliciting reactions, which may have been due to active sensitization.

Dermatitis, Contact↗
PubMed1989

Purification and biochemical characterization of hepatic arylamine N-acetyltransferase from rapid and slow acetylator mice: identity with arylhydroxamic acid N,O-acyltransferase and N-hydroxyarylamine O-acetyltransferase.

An inbred mouse model for the human N-acetylation polymorphism has been used to investigate the biochemical basis for the arylamine N-acetylation polymorphism and the relationship between the cytosolic enzymes arylamine N-acetyltransferase (NAT), arylhydroxamic acid N,O-acyltransferase, and N-hydroxyarylamine O-acetyltransferase. Biochemical studies of partially purified NAT from rapid and slow acetylator mice revealed identical molecular weights of 31,500, activation energies of 21,000 cal/mol, equivalent affinities for acetyl coenzyme A, broad pH optima, the presence of an active site sulfhydryl group, and similar behavior during purification with anion exchange, gel filtration, and hydrophobic interaction chromatography. The enzymes differed in inhibition by hydrogen peroxide and dithiobis(2-nitrobenzoic acid). These observations taken in conjunction with previous investigations indicate that the rapid and slow mouse NAT enzymes are isozymes with minimal structural differences. NATs from rapid and slow acetylator mice were purified more than 10,000-fold by the following sequence of methods: homogenization and fractional centrifugation, protamine sulfate precipitation, and chromatography on DEAE-Trisacryl M, Sephadex G-100, Amethopterin-AH-Sepharose 4B, butyl agarose, and Sephacryl S-200, with a 15-25% recovery. NAT from B6 mice was purified to greater than 95% purity, as judged by silver staining of sodium dodecyl sulfate-polyacrylamide gels. Although only NAT appeared to be subject to a genetic polymorphism as evidenced by N-acetylation activities in liver cytosol, the purified NAT protein possessed arylhydroxamic acid N,O-acyltransferase, N-hydroxyarylamine O-acetyltransferase, and NAT activities. Thus, the cytosolic N-acetyltransferase of mouse liver may catalyze N-, O-, and N,O-acetyltransfer reactions through a common acetylated intermediate of a single protein.

Acetylation↗
PubMed1988

Induction of repair synthesis of DNA in mammary and urinary bladder epithelial cells by N-hydroxy derivatives of carcinogenic arylamines.

Unscheduled DNA synthesis (UDS)-inducing activity was used as a parameter to estimate the abilities of rat mammary epithelial cells and urothelial cells from various species to activate carcinogenic aromatic amine derivatives. The N-hydroxy, N-hydroxy-N-acetyl, N-hydroxy-N-glucuronosyl derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) induced UDS in primary cultures of rat mammary epithelial cells, but 2-AF, the O-glucuronide of N-hydroxy-N-acetyl-2-AF (N-OH-AAF) and 4-ABP did not. Neither the activity of N-OH-AAF, N-hydroxy-N-formyl-2-AF, nor N-acetoxy-N-acetyl-2-AF was significantly altered by paraoxon, an inhibitor of microsomal N-deacetylase. Although N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) also induced UDS, its N-acetyl derivative, which can not be activated by intramolecular, N,O-acetyltransfer, did not. Similarly, rat urothelial cells were responsive to the UDS-inducing activity of this hydroxylamine, but not the hydroxamic acid. In contrast, dog urothelial cells were responsive to both compounds. The UDS-inducing activity of N-OH-AAF was inhibited by paraoxon in the dog, but not in rat urothelial cells. N-Hydroxy-N,N'-diacetylbenzidine induced UDS in the urothelial cells of dog, rat, and rabbit, and a human urothelial cell line, HCV-29, whereas benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine did not. Co-treatment with 12-O-tetradecanoylphorbol-13-acetate did not enable benzidine to induce UDS in dog urothelial cells. Rat mammary epithelial cells activated N-OH-DMABP by acetyl coenzyme A-dependent O-acetylation and N-OH-AAF by N,O-acetyltransfer. They could not N-deacetylate N-OH-AAF. These results suggest that rat mammary and bladder epithelial cells are capable of activating N-arylhydroxylamine metabolites of these carcinogens, probably by N,O-acetyltransfer and O-acetylation, whereas dog urothelial cells are more likely to activate these metabolites by N-deacetylation and a reaction that has yet to be identified.

Aminobiphenyl Compounds↗
PubMed1988

Mutagenicities of N-acyl-N-arylhydroxylamines for Salmonella.

The N-formyl, N-acetyl and N-propionyl derivatives of N-hydroxy-trans-4-aminostilbene (N-OH-AS), N-hydroxy-4-aminobiphenyl (N-OH-ABP) and N-hydroxy-2-aminonaphthalene (N-OH-AN) were synthesized and examined for their mutagenicities in Salmonella typhimurium TA 98. The N-formyl derivatives were direct-acting mutagens possibly due to hydrolysis, either spontaneously or by bacterial enzymes to hydroxylamines. Their mutagenicities were enhanced by rat liver microsomes and cytosol. All acetyl and propionyl derivatives required activation by either liver cytosol or microsomes. NADPH slightly decreased the microsome-mediated mutagenicities of the N-acyl derivatives of N-OH-AN. However, it greatly enhanced the cytosol-mediated mutagenicities of these hydroxamic acids, probably due to stabilization of their hydroxylamine derivatives. The mutagenicities reported here do not correlate with previously reported carcinogenicity data. Thus, data obtained in Salmonella mutagenicity studies may not necessarily directly reflect carcinogenic potential in mammalian systems due to the different mechanisms of activation.

Aminobiphenyl Compounds↗
PubMed1988

Comparison of mutagenesis induced in single- and double-stranded M13 viral DNA by treatment with N-hydroxy-2-aminofluorene.

The specificity of mutagenesis in single-stranded and its complementary double-stranded DNA of the bacteriophage M13mp8 induced by N-hydroxy-2-aminofluorene (N-OH-AF) was analyzed after transfection into its bacterial host Escherichia coli, strain JM103. In this forward mutation assay, randomly modified DNA with increasing levels of aminofluorene (AF) guanine adducts was transfected into competent host JM103 cells with or without prior induction of SOS functions in the host cells. These cells were then screened for mutants of the marker enzyme, beta-galactosidase, on a selective medium. In this assay, the mutation frequency was increased up to 10-fold in host cells with induced SOS functions as compared to the control host cells. Transfection of AF-substituted single-stranded DNA gave a 2.5-fold higher mutation frequency as compared to the double-stranded form at similar levels of AF modification and plaque-forming efficiency. DNA sequence analysis of the mutants showed that AF-modified single- and double-stranded DNA induced base substitutions (52-55%), large deletions (i.e. greater than 300 bp, 25-30%) and frameshifts (16-18%). The mutation sites for 73 base substitutions and frameshifts examined within a limited DNA sequence of 120 bases (6280-6400) were different in single- and double-stranded DNAs. A possible 'hotspot' for base substitutions within one of the two GGCG sequences has also been identified in single-stranded but not double-stranded DNA.

Base Sequence↗