Impact of Cigarette Smoke Condensate on Adhesion-Related Traits and Hemolysin Production of Oral Candida dubliniensis Isolates.

BACKGROUND: Cigarette smoke is associated with higher oral Candida carriage and possible predisposition and increased susceptibility to oral candidal infection. Candida dubliniensis is associated with oral candidosis. Candidal adherence to buccal epithelial cells (BEC) and denture acrylic surfaces (DAS), germ tube (GT) formation, cell surface hydrophobicity (CSH) and hemolysin production are pathogenic traits of Candida. OBJECTIVES: The impact of exposure to cigarette smoke on the aforementioned pathogenic attributes of oral C. dubliniensis has not been studied. Hence, the impact of cigarette smoke condensate (CSC) on adhesion to BEC and DAS, GT formation, CSH and hemolysin production of 20 oral C. dubliniensis isolates after exposure to CSC for 24, 48 and 72 h was ascertained. METHODS: After preparation of the CSC, using an in-house smoking device, the Candida isolates were exposed to the CSC for 24, 48 and 72 h, by a previously described in vitro method. Thereafter, the adhesion to BEC and DAS, GT formation, CSH and hemolysin production of C. dubliniensis isolates was investigated by hitherto described in vitro assays. RESULTS: Exposure to CSC significantly increased the ability of C. dubliniensis oral isolates to adhere to BEC, DAS, GT formation, CSH and produce hemolysin following 24-h, 48-h and 72-h exposure periods to CSC (P < 0.001 for all attributes tested). CONCLUSIONS: Exposure of oral C. dubliniensis isolates to CSC may significantly promote in vitro adhesion traits and hemolysin production of these isolates, thereby augmenting its pathogenicity in vitro in the presence of cigarette smoke.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Casein phosphopeptides and CaCl2 increase penicillin production and cause an increment in microbody/peroxisome proteins in Penicillium chrysogenum.

Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca2+) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca2+ internalization. In this study we observe that the effect of CaCl2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine ß-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin. SIGNIFICANCE: Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca2+ and CPP in the secretory pathway and considering the positive effect that Ca2+ exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl2 addition is very helpful to decipher the processes related to the biosynthesis and secretion of penicillin.

ß1 Tubulin Rather Than ß2 Tubulin Is the Preferred Binding Target for Carbendazim in Fusarium graminearum.

Tubulins are the proposed target of anticancer drugs, anthelminthics, and fungicides. In Fusarium graminearum, ß2 tubulin has been reported to be the binding target of methyl benzimidazole carbamate (MBC) fungicides. However, the function of F. graminearum ß1 tubulin, which shares 76% amino acid sequence identity with ß2 tubulin, in MBC sensitivity has been unclear. In this study, MBC sensitivity relative to that of a parental strain (2021) was significantly reduced in a ß1 tubulin deletion strain but increased in a ß2 tubulin deletion strain, suggesting that ß1 tubulin was involved in the MBC sensitivity of F. graminearum. When strain 2021 was grown in a medium with a low or high concentration of the MBC fungicide carbendazim (0.5 or 1.4 µg/ml), the protein accumulation levels were reduced by 47 and 87%, respectively, for ß1 tubulin but only by 6 and 24%, respectively, for ß2 tubulin. This result was consistent with observations that MBC fungicides are more likely to disrupt ß1 tubulin microtubules rather than ß2 tubulin microtubules in GFP-ß tubulin fusion mutants in vivo. Furthermore, sequence analysis indicated that a difference in tubulin amino acid 240 (240L in ß1 versus 240F in ß2) may explain the difference in MBC binding affinity; this result was consistent with the result that an F240L mutation in ß2 tubulin greatly increased sensitivity to carbendazim in F. graminearum. We suggest that ß1 tubulin rather than ß2 tubulin is the preferred binding target for MBC fungicides in F. graminearum.

Proteomic analysis of an environmental isolate of Rhodotorula mucilaginosa after arsenic and cadmium challenge: Identification of a protein expression signature for heavy metal exposure.

UNLABELLED: A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43±3.34), NaAsO2 (14.76±2.14), CuSO4 (14.73±2.49), and Pb(NO3)2 (15.74±5.3) versus control (7.67±0.95). GSH:GSSG ratio decreased with CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2 and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2. Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast. SIGNIFICANCE: Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as activity of antioxidant enzymes, status of reduced and oxidized glutathione and thiols associated with proteins were all found to be affected by metal exposure. A detailed analysis with arsenic and cadmium pointed to a particular role for arsenic oxidase in arsenic bioaccumulation and tolerance. This is the first time this has been reported in R. mucilaginosa, and suggests that this isolate may have potential in biosorption of these metals in the environment. Proteomic analysis revealed that seven proteins with a variety of roles - ATP synthesis, protein degradation/synthesis, and metabolism of xylose and chitin - were differentially affected by metal exposure in a manner consistent with oxidative stress. These may therefore represent a protein expression signature for exposure to cadmium and arsenic.

Proteomic response of the phytopathogen Phyllosticta citricarpa to antimicrobial volatile organic compounds from Saccharomyces cerevisiae.

Volatile organic compounds (VOCs) released by Saccharomyces cerevisiae inhibit plant pathogens, including the filamentous fungus Phyllosticta citricarpa, causal agent of citrus black spot. VOCs mediate relevant interactions between organisms in nature, and antimicrobial VOCs are promising, environmentally safer fumigants to control phytopathogens. As the mechanisms by which VOCs inhibit microorganisms are not well characterized, we evaluated the proteomic response in P. citricarpa after exposure for 12h to a reconstituted mixture of VOCs (alcohols and esters) originally identified in S. cerevisiae. Total protein was extracted and separated by 2D-PAGE, and differentially expressed proteins were identified by LC-MS/MS. About 600 proteins were detected, of which 29 were downregulated and 11 were upregulated. These proteins are involved in metabolism, genetic information processing, cellular processes, and transport. Enzymes related to energy-generating pathways, particularly glycolysis and the tricarboxylic acid cycle, were the most strongly affected. Thus, the data indicate that antimicrobial VOCs interfere with essential metabolic pathways in P. citricarpa to prevent fungal growth.

Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus.

Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and ß-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-ß-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.

Dodoviscin a inhibits melanogenesis in mouse b16-f10 melanoma cells.

Nowadays, abnormal hyperpigmentation in human skin such as melasma, freckles, and chloasma has become a serious esthetic problem. Cutaneous depigmenting agents could be used to treat these hyperpigmentation-associated dieseases. Dodoviscin A is a natural product isolated from the aerial parts of Dodonaea viscosa. In the present study, we evaluated the effect of dodoviscin A on melanin production in B16-F10 melanoma cells for the first time. We found that dodoviscin A inhibited melanin biosynthesis induced by 3-isobutyl-1-methylxanthine and PD98059 significantly, and there was no obvious effect on the viability of dodoviscin A-treated B16-F10 cells. Meanwhile, dodoviscin A could suppress the activity of mushroom tyrosinase in the cell-free assay system and also decrease 3-isobutyl-1-methylxanthine-induced tyrosinase activity and expression of mature tyrosinase protein in B16-F10 cells. Western blotting analysis showed that dodoviscin A inhibited 3-isobutyl-1-methylxanthine and forskolin-induced phosphorylation of the cAMP response element binding protein in B16-F10 cells. These results indicate that dodoviscin A may be a new promising pigmentation-altering agent for cosmetic and therapeutic applications.

Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans.

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8-Br-cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen-activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non-functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.

Intracellular pH homeostasis plays a role in the tolerance of Debaryomyces hansenii and Candida zeylanoides to acidified nitrite.

The effects of acidified-nitrite stress on the growth initiation and intracellular pH (pH(i)) of individual cells of Debaryomyces hansenii and Candida zeylanoides were investigated. Our results show that 200 microg/ml of nitrite caused pronounced growth inhibition and intracellular acidification of D. hansenii at an external pH (pH(ex)) value of 4.5 but did not at pH(ex) 5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pH(i) decrease at pH(ex) 4.5, suggesting that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For C. zeylanoides, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth. Debaryomyces hansenii was more tolerant to acidified nitrite at pH(ex) 5.5 than C. zeylanoides, as determined by the rate of growth initiation. In combination with the fact that D. hansenii was able to maintain pH(i) homeostasis at pH(ex) 5.5 but C. zeylanoides was not, our results suggest that the ability to maintain pH(i) homeostasis plays a role in the acidified-nitrite tolerance of D. hansenii and C. zeylanoides. Possible mechanisms underlying the different abilities of the two yeast species to maintain their pH(i) homeostasis during acidified-nitrite stress, comprising the intracellular buffer capacity and the plasma membrane ATPase activity, were investigated, but none of these mechanisms could explain the difference.

Farnesol and dodecanol effects on the Candida albicans Ras1-cAMP signalling pathway and the regulation of morphogenesis.

Candida albicans hypha formation which has been stimulated via the Ras1-cAMP-Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl-cAMP. The CAI4-Ras1(G13V) strain, which carries a dominant-active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4-Ras1(G13V) strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP-repressed genes and cells repressed for hypha formation. Because several cAMP-controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1-Cdc35 pathway, thus leading to an alteration of C. albicans morphology.

Effect of copper on acid phosphatase activity in yeast Yarrowia lipolytica.

Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu2+ concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu2+, Co2+, Ni2+, Mn2+ and Mg2+ and inhibited by Ag+ and Cd2+. The most effective ion was Cu2+, especially for the enzyme from cultures in medium containing Cu2+, whereas APase activity in wall-bound fragments was only slightly activated by Cu2+. The content of cellular phosphate involving polyphosphate was decreased by adding Cu2+, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu2+ might depend on derepression of the gene encoding the APase isozyme.

Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis.

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.

Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site.

Histidine (His) tags are one of the most popular fusion tags for the isolation of proteins via metal affinity chromatography. The fusion tag is routinely left attached to the protein when carrying out experiments, with the assumption that the addition has no effect on structure or function. In the present study, we have prepared four proteins of the gene regulatory protein AreA from Aspergillus nidulans for crystallization experiments: a 91-amino acid peptide encompassing the minimal DNA-binding region, both with and without the His-tag (HZFB and ZFB, respectively), and a 155-amino acid protein previously proposed to be the entire DNA-binding domain for AreA, both with and without the His-tag (HG1b and G1b, respectively). To test the integrity of the four AreA proteins, urea denaturation experiments and DNA-binding studies were performed using fluorescence spectroscopy. The DNA-binding data showed similar dissociation constants for all proteins, with Kd values in the nanomolar range. The urea denaturation data, however, clearly indicated that the HZFB protein exhibited a completely different denaturation profile when compared to the ZFB, HG1b, and G1b proteins. The HZFB protein showed a profile indicative of the presence of an altered conformation around the sole tryptophan, whereas the other proteins showed a transition point between 3 and 4 M urea concentration. These data show that, although function was not altered for any of the proteins studied, the structure of one of the His-tagged proteins was different from the native form of that protein.

Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans.

Azoles (imidazoles and triazoles) are used as antifungal agents in agriculture and in medicine, and also for antiestrogen therapy, e.g., for breast cancer treatment. Antifungal activity is based on inhibition of fungal CYP51 (lanosterol 14alpha-demethylase), and estrogen biosynthesis reduction is due to azole inhibition of CYP19 (aromatase). Inhibition of aromatase by antifungal agents is usually an unwanted side effect and may cause endocrine disruption. A fluorimetric assay based on human recombinant CYP19 enzyme with dibenzylfluorescein as a substrate was used to compare the inhibitory potency of 22 azole compounds. Dose responses were established and duplicate datasets were analyzed with a nonlinear mixed-effects model with cumulative normal distribution for the logarithm of concentration. IC50 values (50% inhibitory concentration) of 13 fungicides used in agriculture ranged more than 700-fold, starting from 0.047 microM. The potency of seven human drugs spanned more than 7000-fold, starting from 0.019 microM. Most potent fungicides included prochloraz, flusilazole, and imazalil, and most potent medicinal antifungals were bifonazole, miconazole, and clotrimazole. These in vitro data indicate that the top-ranking azoles used as antifungal agents or drugs are as potent inhibitors of aromatase as are antiestrogen therapeutics used to treat breast cancer. These putative effects of azole agents and drugs on steroid biosynthesis and sex hormone balance should be considered when used in human subjects and also in wildlife exposed to azole fungicides used in agriculture.

Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans.

To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([alpha-(32)P]8-azidoATP; IC(50)=0.76 microM) and drug-substrates ([(3)H]azidopine and [(125)I]iodoarylazidoprazosin; IC(50) approximately 12 microM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 microM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy.

Cremophor EL stimulates mitotic recombination in uvsH//uvsH diploid strain of Aspergillus nidulans.

Cremophor EL is a solubilizer and emulsifier agent used in the pharmaceutical and foodstuff industries. The solvent is the principal constituent of paclitaxel's clinical formulation vehicle. Since mitotic recombination plays a crucial role in multistep carcinogenesis, the study of the recombinagenic potential of chemical compounds is of the utmost importance. In our research genotoxicity of cremophor EL has been studied by using an uvsH//uvsH diploid strain of Aspergillus nidulans. Since it spends a great part of its cell cycle in the G2period, this fungus is a special screening system for the study of mitotic recombination induced by chemical substances. Homozygotization Indexes (HI) for paba and bi markers from heterozygous B211//A837 diploid strain were determined for the evaluation of the recombinagenic effect of cremophor EL. It has been shown that cremophor EL induces increase in mitotic crossing-over events at nontoxic concentrations (0.05 and 0.075% v/v).

Differential sensitivity of plant and yeast MRP (ABCC)-mediated organic anion transport processes towards sulfonylureas.

The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes. Due to broad substrate spectra, specific modulators of these proteins are still lacking. Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor. Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants. To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated. Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein. Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide. Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist. Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.