Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.
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We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans. (+info)
Protection of ebselen against anoxic damage of cultured neurons of cerebral cortex.
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AIM: To study the protective effect of ebselen on anoxic damage of brain cells. METHODS: On d 10 after plating of the cortical neurons from 1-d-old rat, cultures were placed under 95% N2 + 5% CO2 for 2-6 h. Lactate dehydrogenase (LDH) in supernatant, thiobarbituric acid reactive substance (TBARS) and glutathione peroxidase (GSH-Px) activity of neurons were determined. RESULTS: Under anoxia, efflux of LDH and TBARS from cultured neurons increased while GSH-Px activity decreased. Ebselen reduced the efflux of LDH and TBARS in a dose-related manner and increased the total GSH-Px activity. CONCLUSION: Ebselen can protect neurons from anoxic damage. (+info)
Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans.
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Oral infections caused by the yeast Candida albicans are some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. In C. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination of Candida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance. 35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition. (+info)
Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450.
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The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another fluconazole-resistant isolate were homozygotes with respect to CYP51, although the typical wild-type, fluconazole-susceptible C. albicans was a CYP51 heterozygote. These findings suggest that part of the fluconazole-resistant phenotype of C. albicans DUMC136 was acquired through a mutation-prone area of CYP51, an area which might promote the formation of fluconazole-resistant CYP51, along with a mechanism(s) which allows the formation of a homozygote of this altered CYP51 in this diploid pathogenic yeast. (+info)
When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.
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The inclusion of uric acid in the incubation medium during copper-induced low-density lipoprotein (LDL) oxidation exerted either an antioxidant or pro-oxidant effect. The pro-oxidant effect, as mirrored by an enhanced formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances and increase in negative charge, occurred when uric acid was added late during the inhibitory or lag phase and during the subsequent extensive propagation phase of copper-stimulated LDL oxidation. The pro-oxidant effect of uric acid was specific for copper-induced LDL oxidation and required the presence of copper as either Cu(I) or Cu(II). In addition, it became much more evident when the copper to LDL molar ratio was below a threshold value of approx. 50. In native LDL, the shift between the antioxidant and the pro-oxidant activities was related to the availability of lipid hydroperoxides formed during the early phases of copper-promoted LDL oxidation. The artificial enrichment of isolated LDL with alpha-tocopherol delayed the onset of the pro-oxidant activity of uric acid and also decreased the rate of stimulated lipid peroxidation. However, previous depletion of alpha-tocopherol was not a prerequisite for unmasking the pro-oxidant activity of uric acid, since this became apparent even when alpha-tocopherol was still present in significant amounts (more than 50% of the original values) in LDL. These results suggest, irrespective of the levels of endogenous alpha-tocopherol, that uric acid may enhance LDL oxidation by reducing Cu(II) to Cu(I), thus making more Cu(I) available for subsequent radical decomposition of lipid peroxides and propagation reactions. (+info)
Optimized expression and catalytic properties of a wheat obtusifoliol 14alpha-demethylase (CYP51) expressed in yeast. Complementation of erg11Delta yeast mutants by plant CYP51.
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CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14alpha-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C. E. & Halkier, B.A. (1997) Plant J. 11, 191-201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun. 230, 381-385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32-39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum-wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450.(L culture)-1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14alpha-demethylation of obtusifoliol very actively (Km,app = 197 microm, kcat = 1.2 min-1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 microM) and eburicol (Ks = 2.5 microm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 microM), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and gamma-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 microM) and gamma-ketotriazole (0.40 microM) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules. (+info)
Comparison of the toxicity of fluconazole and other azole antifungal drugs to murine and human granulocyte-macrophage progenitor cells in vitro.
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We studied the inhibitory effects on colony formation by granulocyte-macrophage colony forming units (cfu-gm) of eight azole antifungal agents in vitro. All agents, except fluconazole, inhibited colony formation dose-dependently with 50% inhibitory concentrations (IC50) in the range of 0.78-49 micromol/L in cultures of murine and human bone marrow. For human cells, the IC50 values were 0.553 mg/L for itraconazole, 1.24 mg/L for saperconazole, 2.58 mg/L for clotrimazole, 5.33 mg/L for miconazole, 6.17 mg/L for econazole, 6.27 mg/L for ketoconazole and 8.38 mg/L for oxiconazole. The IC50 of itraconazole for human cfu-gm in vitro was similar to the plasma level of this drug recommended for systemic antifungal therapy (>0.5 mg/L) thus indicating the potential clinical relevance of our data. The IC50 of ketoconazole for human cfu-gm in vitro may be exceeded by plasma levels produced in vivo by high (> or =400 mg) doses, whereas fluconazole failed to reduce colony formation by 50% even at 100 mg/L, a concentration not reached in vivo even after extremely high doses (2000 mg/day). To most of the drugs studied, murine progenitor cells seemed to be less sensitive than the human ones. There was, however, a close correlation between the murine and human log IC50 values of the drugs (r2 = 0.964, P< 0.001), suggesting that cultures of murine bone marrow may be suitable to predict the in-vitro toxicity of azole antifungals to human cfu-gm. (+info)
The inhibition of mammalian 15-lipoxygenases by the anti-inflammatory drug ebselen: dual-type mechanism involving covalent linkage and alteration of the iron ligand sphere.
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Mammalian lipoxygenases have been implicated in inflammation and atherosclerosis and, thus, lipoxygenase inhibitors may be of pharmacological interest. In cells, lipoxygenases occur in a catalytically silent ground state that requires activation to become active. We found that the seleno-organic drug ebselen [2-phenyl-1, 2-benzisoselenazol-3(2H)-one], which exhibits anti-inflammatory properties, irreversibly inhibited pure rabbit 15-lipoxygenase, with an IC50 in the nM range when preincubated with the enzyme in the absence of fatty acid substrates. Subsequent dialysis, gel filtration, or substrate addition did not restore the enzyme activity, and experiments with [14C]ebselen indicated a covalent linkage of the drug. The presence of sulfhydryl compounds in the incubation mixture prevented both enzyme labeling and inactivation, but we did not see any reactivation when sulfhydryl compounds were added afterward. X-ray absorption studies indicated that ebselen did alter the geometry of the iron ligand sphere, and the data are consistent with an iron complexation by the drug. When fatty acid substrate was present during lipoxygenase-ebselen interaction, the inhibitory potency was strongly reduced and a competitive mode of action was observed. These data suggest that ebselen inactivated the catalytically silent ground-state lipoxygenase irreversibly by covalent linkage and alteration of the iron ligand sphere. In contrast, it functions as a competitive inhibitor of the catalytically active enzyme species. The pharmacological relevance of ebselen as a potential in vivo lipoxygenase inhibitor will be discussed. (+info)