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S L Kelly

Publications and source records attributed to S L Kelly.

At least 109 records · Page 6Linked to original sources
PubMed1991

Chemostat studies of microsomal enzyme induction in Saccharomyces cerevisiae.

Using cells grown in a chemostat at steady state, the levels of various components of the microsomal electron transport chain of Saccharomyces cerevisiae were examined. Cytochrome P450 haemoprotein levels measured in cells grown in medium with a dissolved oxygen concentration of 15% were induced between 10- and 20-fold over levels in cells grown in medium containing 70% dissolved oxygen concentration. An increase in the dilution rate of a culture growing in medium containing 15% dissolved oxygen resulted in an increase in the residual glucose concentration of the medium. This was paralleled by an increase in the microsomal levels of cytochrome P450. The rise could not be attributed either to increases in the concentration of ethanol in the chemostat or to an increase in the proportion of energy generated using fermentative pathways. However, this effect was not observed in cells grown in an oxygen concentration of 70%. Cytochrome b5 haemoprotein levels were also induced approximately three-fold by reducing the dissolved oxygen concentration from 70% to 15%. Changes in the medium glucose concentration from 0.03% to 1.6% (w/v) had no effect on the levels of this enzyme. Conversely, levels of cytochrome P450 NADPH reductase appeared lower in cells grown in 15% as opposed to 70% dissolved oxygen concentration. Northern slot blot analysis of total RNA extracted from chemostat-grown cells, probed with a C-14 sterol demethylase cytochrome P450 gene (cytochrome P450 LIA1), revealed a pattern of message induction which matched that of the cytochrome P450 haemoprotein, indicating that control of the levels of this enzyme was at least partially transcriptional.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1991

Expression of N-acetyl transferase in a human monocytic cell-line, U937.

1. N-acetyl transferase (NAT) catalyses the acetylation of arylamine and hydrazine drugs and other xenobiotics. The activity of one isozyme (polymorphic NAT) varies amongst individuals but the other (monomorphic NAT) does not. 2. The human monocytic cell-line U937 transcribes the gene for monomorphic N-acetyl transferase. 3. Although the gene for polymorphic N-acetyl transferase is present in these cells, its expression is not detected. 4. It is concluded that U937 cells are a useful model for studying the metabolism of arylamines and hydrazines by human monomorphic N-acetyl transferase.

Acetyltransferases↗
PubMed1990

Interaction of microsomal cytochrome P-450 isolated from Aspergillus fumigatus with fluconazole and itraconazole.

A simple procedure has been developed for isolating a microsomal fraction from Aspergillus fumigatus which contains cytochrome P-450. Maximum absorbance of the carbon monoxide/ferrous cytochrome P-450 difference spectrum was at a wavelength of 451 nm. The triazole antifungal agents, fluconazole and itraconazole, produced type II binding difference spectra with ferric cytochrome P-450 in A. fumigatus microsomes and an azole concentration of 0.5 microM was sufficient to saturate the spectroscopic response when the cytochrome P-450 concentration was 0.07 microM. This extremely high affinity binding precluded the determination of apparent dissociation constants for the cytochrome P-450-azole complexes. Itraconazole was found to have a marginally greater affinity for cytochrome P-450 than fluconazole, as determined from a comparison of their potential to reduce the rate of binding of carbon monoxide to the ferrous haemoprotein. The high affinity binding of both compounds to the cytochrome P-450 from A. fumigatus is consistent with their proposed antifungal mode of action through inhibition of the sterol 14 alpha-demethylase cytochrome P-450 required for ergosterol biosynthesis.

Antifungal Agents↗
PubMed1990

A novel method for studying ergosterol biosynthesis by a cell-free preparation of Aspergillus fumigatus and its inhibition by azole antifungal agents.

A novel procedure has been developed for measuring ergosterol biosynthesis from [14C]mevalonate in a cell-free extract prepared from Aspergillus fumigatus. Ergosterol accounted for approximately 60% of the 4, 14-desmethylated sterol fraction which in turn totalled 13.2% of the non-saponifiable lipid produced. The other major sterol fractions were 4, 4-dimethylated sterols and 4-monomethylated sterols which accounted for 30.8% and 20.1% respectively of non-saponifiable lipid. The cell-free system had a narrow pH optimum of 7.2-7.4 for desmethylated sterol biosynthesis. Activity decreased by 94% at pH 6.5. Fluconazole (10(-4) M), ketoconazole (10(-6) M) and itraconazole (10(-6) M) inhibited formation of desmethylated sterols by greater than 85%, while 4-monomethylated sterols and 4, 4-dimethylated sterols were increased. The IC50s for inhibition of desmethylated sterol biosynthesis were 1.4 x 10(-6) M for fluconazole, 4.0 x 10(-8) M for ketoconazole, and 3.3 x 10(-8) M for itraconazole. The difference in intrinsic potency between fluconazole and ketoconazole is particularly interesting in view of the fact that fluconazole is a more effective agent than ketoconazole in an animal infection model of systemic aspergillosis.

Antifungal Agents↗
PubMed1989

Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals.

Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis. When treated with the sterol 14 alpha-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14 alpha-methyl-erogosta-8,24(28)-dien-3 beta, 6 alpha-diol (14-methyl-3,6 diol). The stringent desaturase mutant, A2, accumulated 14 alpha-methyl-8,24(28)-dien-3 beta-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole. Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation. We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition. We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.

Antifungal Agents↗
PubMed1989

A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

Animals↗
PubMed1989

Genetic and physiological analysis of azole sensitivity in Saccharomyces cerevisiae.

Ketoconazole and fluconazole are azole antifungal agents which inhibit cytochrome P-450 mediated sterol C14 demethylation during ergosterol biosynthesis. We report on the activity of these antifungals on a variety of Saccharomyces cerevisiae strains grown under differing conditions known to affect cyt P-450 levels. Only slight increases in resistance to azoles were observed under conditions which induce the yeast cyt P-450 from undetectable levels. Strain variation was observed, with some strains exhibiting a fungicidal, and others a fungistatic response. Two cyt P-450 deficient mutants examined exhibited resistance to treatment with fluconazole and ketoconazole. This was attributed, at least in part, to an additional defect in sterol delta 5,6 desaturation and possibly to reduced cellular levels of azole drug.

Cytochrome P-450 Enzyme System↗
PubMed1988

Isolation and analysis of ketoconazole resistant mutants of Saccharomyces cerevisiae.

Nine mutants of Saccharomyces cerevisiae which are resistant to ketoconazole, have been isolated and characterized. In each case the mutation is nuclear in origin and allelic to a previously described mutation, erg3, which gives rise to a block in the delta 5-6 desaturation step of ergosterol biosynthesis. The significance of this second site mutation to the point of inhibitory action of ketoconazole, that is the P-450-mediated C-14 demethylation of lanosterol, is discussed.

Cytochrome P-450 Enzyme System↗
PubMed1988

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells.

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

Animals↗
PubMed1986

Genetic effects of methylmethanesulphonate during meiosis in Saccharomyces cerevisiae.

Enhanced mutagenic action after methylmethanesulphonate (MMS) treatment was found in pre-replicative meiotic yeast cells of the strain D7. The level of gene conversion after MMS treatment rose above the spontaneous level during the period of commitment to meiotic recombination, but at later times into meiosis became indistinguishable from the full meiotic level. In contrast reciprocal recombination detected between ade 2 and the centromere of chromosome XV after MMS treatment remained above the spontaneous level up to commitment to meiotic cell division with relatively high levels through meiotic prophase I. These results are discussed in relation to MMS-induced damage and its repair, particularly double-strand break repair.

Gene Conversion↗
PubMed1985

Differences in the cytochrome P-450 enzymes of sterol C-14 demethylase mutants of Saccharomyces cerevisiae.

A number of nystatin-resistant strains of S. cerevisiae have been isolated which are defective in lanosterol C-14 demethylation, a reaction normally catalysed by cytochrome P-450. In this paper two of these strains have been compared and found to have differences in their reduced-CO difference spectra indicating different distortions in the enzyme molecule. Nystatin resistance in the C-14 demethylation deficient SG1 in shown to be determined by a single gene, and a sterol 5,6-desaturase defect does not appear to be required for viability of SG1, was reported for the C-14 demethylase deficient isolate JR4 by Taylor et al. (1983). There are at least two discernable mutant phenotypes for the yeast cytochrome P-450 structural gene which give a C-14 demethylase defect.

Cytochrome P-450 Enzyme System↗
PubMed1985

Interaction between yeast cytochrome P-450 and chemical carcinogens.

The binding of various chemical carcinogens to the glucose-inducible form of cytochrome (cyt.) P-450 in Saccharomyces cerevisiae was examined with reference to known metabolism in mammalian systems. All the carcinogens examined gave spectral interactions with the yeast cytochrome. The carcinogens benzo[a]pyrene and cyclophosphamide gave Type I binding, while benzidine and 2-naphthylamine gave Type II binding spectra indicating binding at the haem group. Dimethylnitrosamine gave undetectable binding in yeast microsomal fractions, but a Type I binding spectrum was seen with purified cyt. P-450 preparations. The demonstration that these carcinogens bind to yeast cyt. P-450 is discussed in the light of their genotoxicity.

2-Naphthylamine↗
PubMed1983

Cyclic variations in sensitivity to X-irradiation during meiosis in Saccharomyces cerevisiae.

Cyclic variation in mutation induction and lethality was found following X-irradiation during meiosis in Saccharomyces cerevisiae. An enhanced mutagenic response was found in meiotic G1 phase cells in comparison to cells later in meiosis, similar to the response shown during mitosis, but meiotic G1 phase cells appeared more resistant to the lethal effects of X-irradiation than mitotic G1 phase cells. Resistance to the lethal effects of X-rays was found during meiotic DNA synthesis in the strain SK1, which may indicate the operation of a sister-chromatid exchange repair mechanism. A difference was found between gene conversion which appeared to be at a maximum by the end of meiotic DNA synthesis and reciprocal recombination, which could be induced up to prophase I.

DNA Repair↗
PubMed1983

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Meiosis↗
PubMed1983

N-methyl-N'-nitro-N-nitrosoguanidine induced genetic change during the meiotic cell cycle in Saccharomyces cerevisiae: an absence of S-phase specificity.

In the yeast Saccharomyces cerevisiae enhanced nuclear mutagenesis was found during meiotic DNA synthesis after treatment with N-methyl-N'-nitrosoguanidine (MNNG), but not during meiotic DNA synthesis. Further experiments found no significant variation in the mol. wt. of DNA from control or MNNG-treated samples during meiosis after alkaline sucrose gradient analysis. Using tritiated MNNG no variation in uptake of MNNG by meiotic cells was found, but enhanced binding of label from [3H]MNNG to mitochondrial DNA was detected during meiotic DNA synthesis when mitochondrial DNA synthesis occurred. In contrast, no enhanced binding of label from [3H]MNNG to the nuclear DNA during meiotic DNA synthesis was found.

DNA Replication↗