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The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.
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Pollutants, such as polycyclic aromatic hydrocarbons (PAHs), e.g., benzo(a)pyrene (BaP), are common components of contaminating mixtures. Such compounds are ubiquitous, extremely toxic, and they pollute soils and aquatic niches. The need for new microorganism-based remediation strategies prompted researchers to identify the most suitable organisms to eliminate pollutants without interfering with the ecosystem. We analyzed the effect caused by BaP on the growth properties of Candida albicans, Debaryomyces hansenii, Rhodotorula mucilaginosa, and Saccharomyces cerevisiae. Their ability to metabolize BaP was also evaluated. The aim was to identify an optimal candidate to be used as the central component of a mycoremediation strategy. The results show that all four yeast species metabolized BaP by more than 70%, whereas their viability was not affected. The best results were observed for D. hansenii. When an incubation was performed in the presence of a cytochrome P450 (CYP) inhibitor, no BaP degradation was observed. Thus, the initial oxidation step is mediated by a CYP enzyme. Additionally, this study identified the D. hansenii DhDIT2 gene as essential to perform the initial degradation of BaP. Hence, we propose that D. hansenii and a S. cerevisiae expressing the DhDIT2 gene are suitable candidates to degrade BaP in contaminated environments.
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The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 µg·mL-1; EPL produced a K+ and Ca2+ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.
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Antifúngicos , Candida albicans , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/metabolismo , Testes de Sensibilidade Microbiana , Polilisina/metabolismo , Polilisina/farmacologia , Saccharomyces cerevisiae/metabolismoRESUMO
Sarcopenia is a syndrome that leads to physical disability and that deteriorates elderly people´s life quality. The etiology of sarcopenia is multifactorial, but mitochondrial dysfunction plays a paramount role in this pathology. Our research group has shown that the combined treatment of metformin (MTF) and exercise has beneficial effects for preventing muscle loss and fat accumulation, by modulating the redox state. To get an insight into the mechanism of the combined treatment, the mitochondrial bioenergetics was studied in the mitochondria isolated from old female Wistar rats quadriceps muscles. The animals were divided into six groups; three performed exercise on a treadmill for 5 days/week for 20 months, and the other three were sedentary. Also, two groups of each were treated with MTF for 6 or 12 months. The rats were euthanized at 24 months. The mitochondria were isolated and supercomplexes formation along with oxygen consumption, ATP synthesis, and ROS generation were evaluated. Our results showed that the combined treatment for 12 months increased the complex I and IV activities associated with the supercomplexes, simultaneously, ATP synthesis increased while ROS production decreased, indicating a tightly coupled mitochondria. The role of exercise plus the MTF treatment against sarcopenia in old muscles is discussed.
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Metformina , Sarcopenia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Idoso , Animais , Metabolismo Energético , Feminino , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Músculo Esquelético/fisiologia , Músculo Quadríceps/patologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologiaRESUMO
Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.
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The authors have retracted this article [1]. After publication the dye used in this study was analysed by NMR and mass spectroscopy and found not to be acridine yellow, but rather, was identified as thioflavin T.
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Translocation of ions and other molecules across the plasma membrane of yeast requires the electric potential generated by a H+-ATPase. We measured under different conditions fluorescence changes and accumulation of acridine yellow, looking for qualitative and quantitative estimations of the PMP in Saccharomyces cerevisiae in various conditions. Fluorescence changes indicated an accumulation of the dye requiring a substrate, and accumulation and quenching by mitochondria that could be released by an uncoupler. K+ produced a decrease of the fluorescence that was much lower upon the addition of Na+. These changes were confirmed by images of the cells under the microscope. The dye accumulation under different conditions showed changes consistent with the physiological situation of the cells. Since it accumulates due to the PMP, but a large part of it binds to the internal components, we permeabilized the cells with chitosan to subtract this factor and correct the accumulation data. Both raw and corrected values of PMP are different to those obtained before by other authors and our group, showing acridine yellow as a promising indicator to follow changes of the PMP by the fluorescence changes, but also by its accumulation. Under conditions described, the dye is a low cost monitor to define and follow qualitative and quantitative changes of PMP in yeast. Acridine yellow can also be used to follow changes of the mitochondrial membrane potential.
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Aminoacridinas/análise , Membrana Celular/fisiologia , Potenciais da Membrana , Potencial da Membrana Mitocondrial , Métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae/citologiaRESUMO
The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 µ g·mL(-1) produced (a) an efflux of K(+) and stimulation of extracellular acidification, (b) an inhibition of Rb(+) uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca(2+). It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K(+), it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca(2+), (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL(-1) are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells.
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Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Quitosana/farmacologia , Cálcio/metabolismo , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quitinases/metabolismo , Contagem de Colônia Microbiana , Ensaios Enzimáticos , Fermentação/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Nucleotídeos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfatos/metabolismo , Potássio/metabolismo , Potássio/farmacologia , Rubídio/metabolismo , Eletricidade EstáticaRESUMO
The effects of ketoconazole and miconazole uptake on K(+) transport and the internal pH of Saccharomyces cerevisiae were studied. The uptake of both drugs was very fast, linear with concentration and not dependent on glucose, indicating entrance by diffusion and concentrating inside. Low (5.0µM) to intermediate concentrations (40µM) of both drugs produced a glucose-dependent K(+) efflux; higher ones also produced a small influx of protons, probably through a K(+)/H(+) exchanger, resulting in a decrease of the internal pH of the cells and the efflux of material absorbing at 260nm and phosphate. The cell membrane was not permeabilized. The K(+) efflux with miconazole was dependent directly on the medium pH. This efflux results in an increased membrane potential, responsible for an increased Ca(2+) uptake and other effects. These effects were not observed with two triazolic antifungals. A decrease of the Zeta (ζ) potential was observed at low concentrations of miconazole. Although the main effect of these antifungals is the inhibition of ergosterol synthesis, K(+) efflux is an important additional effect to be considered in their therapeutic use. Under certain conditions, the use of single mutants of several transporters involved in the movements of K(+) allowed to identify the participation of several antiporters in the efflux of the cation.
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Homeostase/efeitos dos fármacos , Cetoconazol/química , Miconazol/química , Potássio/química , Saccharomyces cerevisiae/efeitos dos fármacos , Antifúngicos/farmacologia , Cátions , Fluconazol/farmacologia , Glucose/química , Glucosefosfato Desidrogenase/metabolismo , Concentração de Íons de Hidrogênio , Itraconazol/farmacologia , Nucleotídeos/química , Consumo de Oxigênio , PrótonsRESUMO
In this study, amiodarone, at very low concentrations, produced a clear efflux of K(+). Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K(+) efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca(2+) and H(+), the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca(2+) concentration, as well as the decreased internal pH. The Deltatok1 and Deltanha1 mutations resulted in a smaller effect of amiodarone, and Deltatrk1 and Deltatrk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K(+), through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.
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Amiodarona/farmacologia , Antifúngicos/farmacologia , Cálcio/metabolismo , Hidrogênio/metabolismo , Potássio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacosRESUMO
Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.