Biomedical subjects

S L Kelly

Publications and source records attributed to S L Kelly.

At least 55 records · Page 3Linked to original sources

PubMed1998

Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains.

From each of two AIDS patients with oropharyngeal candidiasis, five Candida albicans isolates from recurrent episodes of infection which became gradually resistant against fluconazole during antimycotic treatment were analyzed for molecular changes responsible for drug resistance. In both patients, a single C. albicans strain was responsible for the recurrent infections, but the CARE-2 fingerprint pattern of the isolates exhibited minor genetic alterations, indicating that microevolution of the strains took place during fluconazole therapy. In the isolates from patient 1, enhanced mRNA levels of the MDR1 gene, encoding a multiple drug resistance protein from the superfamily of major facilitators, and constitutive high expression of the ERG11 gene, coding for the drug target enzyme sterol 14alpha-demethylase, correlated with a stepwise development of fluconazole resistance. The resistant strains exhibited reduced accumulation of fluconazole and, for the last in the series, a slight increase in drug needed to inhibit sterol 14alpha-demethylation in vitro. In the isolates from patient 2, increased MDR1 mRNA levels and the change from heterozygosity to homozygosity for a mutant form of the ERG11 gene correlated with continuously decreased drug susceptibility. In this series, reduced drug accumulation and increased resistance in the target enzyme activity, sterol 14alpha-demethylase, were observed. These results demonstrate that different molecular mechanisms contribute to a gradual development of fluconazole resistance in C. albicans.

AIDS-Related Opportunistic Infections↗

PubMed1998

NADPH cytochrome P-450 oxidoreductase and susceptibility to ketoconazole.

The phenotype of a strain of Saccharomyces cerevisiae containing a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system in cpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.

Antifungal Agents↗

PubMed1998

Movement hyperacuity in childhood amblyopia.

BACKGROUND: Amblyopia results in deficits in a number of visual functions in both the amblyopic and dominant eye. The present work describes oscillatory movement displacement thresholds (OMDT) in childhood amblyopia. METHODS: The OMDT from the dominant and amblyopic eyes of 50 orthoptic patients (aged 74 (SD 16) months) were compared with those from a group of 24 controls (79 (21) months). OMDT were measured using a forced choice staircase procedure. Subjects were asked to identify which of the computer controlled monitors displayed the oscillating stimulus. Visual acuity and stereoscopic responses were noted from clinical records. RESULTS: Amblyopic children demonstrating stereopsis showed no significant OMDT deficit in the amblyopic eye. Those children having no stereopsis had elevated OMDT in the amblyopic eye (p < 0.05). Results suggest that the dominant eye of children with amblyopia may also have a pattern of visual development which is anomalous (difference in correlation coefficient with age; p < 0.05). CONCLUSION: OMDT deficits demonstrated in some amblyopic eyes indicate that amblyopia is incompletely described by its "clinical" definition. Results suggest that the dominant eye in those with unilateral amblyopia may not be "normal".

Aging↗

PubMed1998

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells.

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.

Animals↗

PubMed1997

Sterol 22-desaturase, cytochrome P45061, possesses activity in xenobiotic metabolism.

CYP61 was revealed in the sequencing of the yeast genome on chromosome XIII and was the last member of the CYP superfamily in yeast to be discovered. We show here that besides the housekeeping role in 22-desaturation during ergosterol biosynthesis the enzyme is also that responsible for benzo(a)pyrene metabolism/promutagen activation by yeast in genotoxicity assays. This enzyme may represent an ancestral activity for the superfamily which allowed xenobiotic metabolism for the first time.

Benzo(a)pyrene↗

PubMed1997

Molecular analysis of cyp51 from fluconazole-resistant Candida albicans strains.

The target enzyme for fluconazole is sterol 14 alpha-demethylase, a cytochrome P450 encoded by cyp51. One mechanism of fluconazole resistance likely to occur in Candida albicans is through an altered target site. To test this hypothesis DNA sequencing of the cyp51 coding sequence from 19 fluconazole-resistant and 19 fluconazole-sensitive C. albicans was undertaken. A number of point mutations were identified in the resistant isolates which were not present in the sensitive ones: F105L (five), E266D (five), K287R (one), G448G (one), G450E (one), G464S (three) and V488I (one). These alterations are discussed in the light of a molecular model of the enzyme regarding potential roles in resistance. It was also demonstrated that sequence-specific primers can be employed to identify polymorphisms which may be associated with resistance; diagnostic tests for resistant strains will prove of value in combating this serious clinical problem.

Antifungal Agents↗

PubMed1997

Characterization of Saccharomyces cerevisiae CYP61, sterol delta22-desaturase, and inhibition by azole antifungal agents.

Cytochrome P-45061 (CYP61) was a cytochrome P-450 revealed during the yeast genome project when chromosome XIII was sequenced. Here we report on the properties of this second microsomal P-450 of vegetatively growing yeast. The enzyme kinetics associated with its endogenous role in sterol Delta22-desaturation revealed a Km of 20.4 microM and a Vmax of 2.9nmol/min/nmol CYP61. The affinity of the enzyme for antifungal drugs was characterized to investigate its potential role in determining tolerance to these sterol 14alpha-demethylase (CYP51) inhibitors. Drug binding induced a type II spectral change, which became saturated at equimolar concentrations of azole drug and P-450. Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement. These and Ki determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole drugs when compared with data available for CYP51, and the implications for antifungal treatment were considered.

Antifungal Agents↗

PubMed1997

Differential inhibition of Candida albicans CYP51 with azole antifungal stereoisomers.

Azole antifungal compounds are important in agriculture and in the treatment of mycotic infection. The target enzyme, sterol 14 alpha-demethylase (CYP51), is inhibited through binding of triazole N-4 to the haem of this P450, as a sixth ligand together with the N-1 substituent groups interacting in some way with the apoprotein. Here we use Saccharomyces cerevisiae expression systems for the target enzyme of Candida albicans to investigate binding of enantiomers of the azole antifungal compounds SCH39304 and tetraconazole. A molecular model produced previously provided qualitative explanations for these differences. Interaction of the azole antifungal aromatic group with Phe-233 or -235 may cause the higher activity for (R)-tetraconazole while inactivity of the (SS)-enantiomer of SCH39304 was predicted to result from incompatibility of the hydrophilic sulfonyl moiety when located into the hydrophobic pocket of the active site.

Antifungal Agents↗

PubMed1997

The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.

Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.

Amino Acid Sequence↗

PubMed1997

Reduced intracellular accumulation of azole antifungal results in resistance in Candida albicans isolate NCPF 3363.

Candida albicans strain NCPF 3363 was isolated from a British patient with chronic mucocutaneous candidiasis (CMC) and confirmed to be resistant to azole antifungal compounds. In this study we investigate the molecular basis of resistance and show that azole tolerance in NCPF 3363 was associated with reduced intracellular accumulation of drug and not reduced affinity for the target site, as previously indicated. Relative impermeability or the presence of transporters related to those responsible for multidrug resistance are implicated in the mechanism of resistance.

Antifungal Agents↗

PubMed1997

Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation.

Fluconazole resistance occurs in > 10% of cases of candidosis during the late stages of AIDS. We show here in two clinical isolates that resistance was caused by defective sterol delta5,6-desaturation. This altered the type of sterol accumulating under fluconazole treatment from 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha -diol to 14alpha-methylfecosterol which is capable of supporting growth. A consequence of this mechanism of azole resistance is that an absence of ergosterol causes cross-resistance to the other major antifungal agent available, amphotericin B. The results also show that growth arrest after fluconazole treatment of C. albicans in clinical conditions is caused by 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha -diol accumulation.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Cytochrome P450 3A4 mediated metabolism of 2,4-dichlorophenol.

The metabolism of the environmental pollutant and hepatocarcinogen 2,4-dichlorophenol (2,4-DCP) was studied using microsomal fractions and whole-cells of Saccharomyces cerevisiae containing human cytochrome P450 3A4. 2,4-DCP exhibited a typical type I substrate binding spectrum with a K, of 75 microM. 2,4-DCP was metabolised into two major metabolites identified as 2-chloro-1,4-hydroxyquinone and 2-chloro-1,4-benzoquinone in microsomal fractions and whole cells of yeast expressing human cytochrome P450 3A4. A further metabolite, 1,2,4-hydroxybenzene, was also detected during biotransformation by whole cells, but was not observed in microsomal fractions. 2,4-DCP metabolism was dependent on NADPH in microsomal fractions and no activity was observed in microsomal fractions or whole cells of control transformants. Metabolites were identified by TLC followed by GC-MS.

Anthelmintics↗

PubMed1997

Stereoselective interaction of SCH 39304, a triazole, with sterol 14alpha-demethylase of Aspergillus fumigatus.

The inhibitory activity of SCH 39304 and its enantiomers on radial growth and on the target enzyme, sterol 14alpha-demethylase, in Aspergillus fumigatus was studied to assess the role of stereochemistry in the efficacy of the drug. SCH 39304 and the RR(+) enantiomer were active in inhibiting the growth while no inhibition in the growth was observed with the SS(-) enantiomer. The MIC of SCH 39304 for the growth was about twice that of the RR(+) enantiomer. The differences in IC50s of SCH 39304 and its enantiomers for cell-free ergosterol biosynthesis correlated with their variations in MICs and type II binding spectra indicated the SS(-) enantiomer failed to bind to microsomal P450. These results show that the difference between SS(-) and RR(+) enantiomers in interacting with the target enzyme is the cause for significant difference in the potency between these two forms.

Antifungal Agents↗

PubMed1997

In-vitro activity of D0870, a new triazole antifungal drug, in comparison with fluconazole and itraconazole against Aspergillus fumigatus and Candida krusei.

The activity of the new triazole antifungal D0870 was compared with those of itraconazole and fluconazole against Candida krusei and Aspergillus fumigatus, two fungi showing inherent tolerance of fluconazole. The activity of D0870 resembled that of itraconazole against whole cells of C. krusei, but it was less effective against A. fumigatus. However, the effect on sterol biosynthesis, in terms of the sterol type accumulating and IC50 for in-vitro biosynthesis, appeared similar in both species. The superior antifungal effect of D0870 over fluconazole appeared related to better inhibition of ergosterol biosynthesis in A. fumigatus, but in C. krusei this did not account for the entire difference which may result mainly from reduced efflux of drug.

Antifungal Agents↗

PubMed1997

Fluconazole tolerance in clinical isolates of Cryptococcus neoformans.

Eleven isolates of Cryptococcus neoformans were investigated to determine the biochemical basis of their tolerance to fluconazole. The MICs of fluconazole for three isolates with low-level resistance were 3- to 6-fold higher than those for sensitive isolates, while the MICs for four isolates with high-level resistance were 100- to 200-fold higher than those for sensitive isolates. The level of ergosterol present in the isolates varied, and those which had relatively low levels of ergosterol were resistant to amphotericin B. Changes in the affinity of the target enzyme (sterol 14alpha-demethylase) and decreases in the cellular content of fluconazole seemed to be responsible for the resistance in isolates with low-level and high-level resistance, respectively.

Amphotericin B↗

PubMed1997

Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

Azoles↗

PubMed1997

Itraconazole resistance in Aspergillus fumigatus.

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Adult↗

PubMed1997

Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans.

We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.

Antifungal Agents↗