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J C Loper

Publications and source records attributed to J C Loper.

At least 37 records · Page 2Linked to original sources
PubMed1987

Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis.

The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a lambda gt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were induced by alkane to levels 500 to 1000 fold over those detected in glucose-grown cells. The nucleotide sequence of the 3' portion of this gene revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Alkanes↗
PubMed1987

Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae.

We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen. The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain. The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily. These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI. The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam. In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc. Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions. The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.

Amino Acid Sequence↗
PubMed1986

Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae.

We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14 alpha-demethylase. Two plasmids were isolated which transformed yeast to both increased resistance to Kc and increased levels of total P-450. Hybrid-selection and immunoprecipitation experiments showed that these plasmids, pVK1 and pVK2, contained the structural gene for an S. cerevisiae P-450. This conclusion was confirmed by the nucleotide sequence of a portion of pVK2, which revealed an open reading frame encoding a characteristic P-450 heme-binding region.

Amino Acid Sequence↗
PubMed1985

Metabolic activation of 3-(2-chloroethoxy)-1,2-dichloropropene: a mutagen structurally related to diallate, triallate, and sulfallate.

3-(2-Chloroethoxy)-1,2-dichloropropene (CP), a Salmonella promutagen that was recently isolated from a sample of residue organics previously concentrated from drinking water, is structurally related to three other chlorinated promutagens, the S-chloroallyl thiocarbamate herbicides diallate, triallate, and sulfallate. These four chloroallyl ether compounds were found to be similar with respect to strain specificity, potency, and requirement for specific metabolic activation. The 9,000g supernatant (S9) fractions from polychlorinated biphenyl Aroclor 1254- or phenobarbital-induced rats metabolized the four chloroallyl ethers to mutagenic products, whereas S9 from 3-methylcholanthrene-induced or uninduced rats did not. The metabolic activation of CP, diallate, and triallate to mutagens was catalyzed by the 100,000g microsomal pellet of S9 alone, but the activation of sulfallate to mutagenic metabolites required both microsomal and cytosolic fractions of S9. Direct-acting (minus S9) mutagenic metabolites of diallate and triallate could be extracted into methylene chloride from S9 incubation mixtures. Incubations containing S9 and either sulfallate or CP did not yield methylene chloride-extractable metabolites with direct-acting mutagenic activity. On the basis of these results and those from previous studies on the metabolism of diallate, triallate, and sulfallate, a tentative model for the metabolic activation of CP is proposed in which this chloroallyl ether undergoes alpha-carbon hydroxylation to form multiple mutagenic products.

Allyl Compounds↗
PubMed1985

Water contamination and environmental mutagens.

Citizens of industrialized and developing nations share a common concern for safe water sources, but each group must contend with different priorities and problems. Examples of pollution involving surface, ground, and irrigation water are presented. The Salmonella mutagenesis assay has proven to be a valuable bioassay for detection and isolation of unknown water-borne mutagens, and is useful in monitoring the levels of mutagenic pesticides.

Biological Assay↗
PubMed1984

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens.

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.

Allyl Compounds↗
PubMed1983

Effects of metyrapone on microsomal-dependent Salmonella mutagenesis. Studies with chloroallyl ethers and model compounds.

Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone, MTP) is used as an inhibitor of cytochrome P-450 enzymes, particularly those induced by phenobarbital (PB). We examined the effects of MTP on the microsomal dependent mutagenesis of a newly isolated promutagen, 3-(2-chloroethoxy)-1,2-dichloropropene (CP), three S-chloroallyl thiocarbamate herbicides, and four model promutagens aflatoxin B1 (AFB), 2-acetylaminofluorene (2AAF), 2-aminoanthracene (2AA) and benzo[a]pyrene (BP). Salmonella tester strains TA98, TA100 and TA1535 and liver microsomal preparations (S9) from rats induced with PB or Aroclor 1254 (PCB) were employed. For statistical analysis, mutagenesis data were transformed and subjected to two-way analysis of variance. Metyrapone alone was not mutagenic in the absence or presence of S9. In a dose-dependent manner, MTP inhibited mutagenesis of AFB for strains TA98 and TA100 and enhanced mutagenesis of 2AAF, 2AA and BP for these strains. 3-(2-Chloroethoxy)-1, 2-dichloropropene and the herbicides diallate, triallate and sulfallate are all chloroallyl ethers. They are similar in their mutagenesis for Salmonella with respect to strain specificity, mutagenic potency, and requirement for activation by specifically-induced microsomes. Metyrapone inhibited the mutagenesis of CP, triallate and sulfallate for strain TA100 in the presence of either PB- or PCB-induced S9, and had no apparent effect on diallate mutagenesis; the same results were obtained for TA1535 with PCB-induced S9. On this basis, the mutagenic activation of diallate appears to be different from that of the other chloroallyl ethers tested. Our results indicate that MTP can inhibit as well as enhance microsomal dependent mutagenesis for Salmonella. We conclude that MTP may be a useful tool in characterizing pathways for promutagen activation.

Allyl Compounds↗
PubMed1980

Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene.

The synthesis and mutagenicity of two derivatives of 7,12-dimethylbenz[a]anthracene (DMBA; 1), i.e., 1,2-H2DMBA (4) and 1,2,3,4-H4DMBA (5), are reported. These analogues (4 and 5) represent dihydro and tetrahydro A-ring reduced forms of DMBA, a region in the parent hydrocarbon (1) proposed to be involved in metabolism to the ultimate carcinogen. The synthesis for 4 without isolation of intermediates from the tosylhydrazone of 1,2,3,4-tetrahydrobenz[a]anthracene-4,7,12-trione (10) by successive reaction with 8 molar equiv of CH3Li, HI, and NaBH4 represents a novel approach to this hydrocarbon now available in sufficient quantity for biological studies. Interestingly, both of these reduced analogues 4 and 5 exhibited mutagenic activity in the Ames assay in the presence or absence of microsomal activation for strains TA98 and TA100. In these strains, DMBA was active only in the presence of S-9 fraction. In the plasmid-deficient strain TA1537, only tetrahydro analogue 5 exhibited mutagenic activity both in the absence and presence of S-9 fraction.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1980

Malignant transformation of BALB/3T3 cells by residue organic mixtures from drinking water.

Malignant transformation of BALB/3T3 1-13 cells results following treatment with complex mixtures of residue organic chemicals from drinking water samples of five U. S. cities. The U. S. Environmental Protection Agency provided the residues which were obtained by liquid-liquid solvent extraction, and sorption-desorption on XAD-2 resin of samples concentrated by reverse osmosis from drinking water of Philadelphia, PA; New Orleans, LA; Miami, FL; Seattle, WA; and Ottumwa, IA. Transformation was observed using a focus formation assay following 72-hr exposure of cells to residues from one or more samples from each city. Malignancy of several transformed and normal clones was evaluated in athymic (nu/nu) mice. The usefulness of such a transformation assay in detecting potential carcinogenic compounds contained in complex mixtures and the evaluation of possible health effects of residue organics in drinking water are discussed.

Animals↗
PubMed1979

Non-mutagenicity for Salmonella of the chlorinated hydrocarbons aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone.

A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-trichlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlorobenzene were shown to differ from S9 of either control or Aroclor 1254-induced rats in the capacity to activate 2-aminoanthracene mutagenesis.

Aroclors↗
PubMed1977

Histidinol dehydrogenase from salmonella typhimurium and Escherichia coli. Purification, some characteristics and the amino acid sequence around a reactive thiol group.

The purification and some physical properties of histidinol dehydrogenase, L-histidinol-nicotinamide adenine dinucleotide oxido-reductase (EC 1.1.1.23) from either Salmonella typhimurium or Escherichia coli are reported in this paper. Modification of histidinol dehydrogenase with one equivalent of N-(4-dimethylamino-3,5-dinitrophenyl)maleimide at pH 6.8 yields an enzyme that is inactive toward the oxidation of L-histidinol. The modified cysteine residue was located in an acid insoluble tryptic core. The amino acid sequence around the reactive thiol group in S. typhimurium is: Leu-Cys-Gly-Val-Glu-Glu-Ile-Phe, and in E. coli is: Leu-Cys-Gly-Val-Glu-Asp-Val-Phe. These unique sequences show no homology to the reactive thiol groups from some other dehydrogenases.

Alcohol Oxidoreductases↗
PubMed1977

Mutagenic studies of folic acid antagonists.

Compounds that compete with folic acid (folic acid antagonists [FAAs]) become limited in their usefulness in the treatment of leukemia, malaria, and bacterial infections by the rapid development of resistance. Assays of the plasma levels of certain of these FAAs led to the observation, in about 25% of the determinations, that a higher density of growth of Streptococcus faecium var. durans (ATCC 8043) was obtained at an FAA concentration just below the completely inhibitory level than at one-half this concentration. This and other considerations suggested that FAAs may act not only as selective agents for resistant organisms but also as mutagens. Seven FAAs including amethopterin, pyrimethamine, trimethoprim, chlorguanide triazine, an experimental quinazoline, WR-158,122, and two experimental triazines, WR-99,210 and WR-38,839, were tested for mutagenicity in the Salmonella reversion assay developed by Ames et al. (1975). All were found to be negative for strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without microsomal activation. These compounds were then tested as mutagens for three traits in the folic acid-requiring S. faecium. FAAs were shown to cause mutations to folic acid independence, rifampin resistance, and FAA resistance. It is postulated that the FAAs induce mutations by causing thymine deprivation in the folic acid-requiring host.

Culture Media↗
PubMed1976

Resistance plasmids of Pseudomonas aeruginosa: change from conjugative to nonconjugative in a hospital population.

Properties of a population of carbenicillin- and gentamicin-resistant, tobramycin-susceptible Pseudomonas aeruginosa at Veteran's Administration Hospital, Cincinnati, Ohio, have been followed during a 16-month period. As originally described, the strains were isolated from patients with urinary tract colonizations and were predominantly Parke-Davis immunotype 7. For the majority of these organisms, antibiotic resistance was correlated with the presence of a self-conjugative plasmid of incompatibility group P-2. The source and relative incidence of multiply resistant isolates have remained constant during the current study, but the immunotype has shifted form type 7 to type 2. Concomitantly, the population has lost the property of conjugative transfer of resistance, and resistant strains are now compatible with P-2 plasmids. A group P-2 R plasmid, pMG5, will mobilize resistance markers, demonstrating that the multiple resistance of the nonconjugative strains is mediated by R plasmids. Additionally, gentamicin resistance due to either conjugative or nonconjugative plasmids is correlated with the presence of similar gentamicin acetyltransferase activity. pMG5-mobilized plasmids are shown to be incompatible with pMG5. pMG5 is also shown to mobilize resistance markers from nontransferring antibiotic-resistant strains representing populations from Parkland Memorial Hospital, Dallas, Texas, and Cleveland Clinic Foundation, Cleveland, Ohio.

Acetyltransferases↗
PubMed1975

Pseudomonas aeruginosa R factors determining gentamicin plus carbenicillin resistance from patients with urinary tract colonizations.

R factors determining multiple resistance including both gentamicin and carbenicillin have been identified in high incidence among hospital isolates of Pseudomonas aeruginosa. The factors are readily transmitted to other P. aeruginosa but not to Escherichia coli strains K-12 or C, or to Proteus mirabilis. R factor-containing isolates are predominantly immunotype 7 isolated from urinary sources.

Carbenicillin↗
PubMed1975

RPL11, an R factor of Pseudomonas aeruginosa determining carbenicillin and gentamicin resistance.

R factor RPL11 from Pseudomonas aeruginosa determines multiple drug resistance including resistance to gentamicin and carbenicillin. The host range and incompatibility properties of RPL11 are those of incompatibility group P-2. Strains harboring the factor are not altered with respect to the major immunotypes 1 through 7 of Parke-Davis, or with respect to pyocin type by using the 18 indicators of Jones and co-workers. Analytical ultracentrifugation of crude extracts of R factor-containing strains shows a band of satellite DNA with a buoyant density of 1.717 g/cm(3).

Carbenicillin↗