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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.
Assuntos
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.
Assuntos
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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The emergence of microbes resistant to common antibiotics represent a current treat to human health. It has been recently recognized that non-antibiotic labeled drugs may promote antibiotic-resistance mechanisms in the human microbiome by presenting a secondary antibiotic activity; hence, the development of computer-assisted procedures to identify antibiotic activity in human-targeted compounds may assist in preventing the emergence of resistant microbes. In this regard, it is worth noting that while most antibiotics used to treat human infectious diseases are non-peptidic compounds, most known antimicrobials nowadays are peptides, therefore all computer-based models aimed to predict antimicrobials either use small datasets of non-peptidic compounds rendering predictions with poor reliability or they predict antimicrobial peptides that are not currently used in humans. Here we report a machine-learning-based approach trained to identify gut antimicrobial compounds; a unique aspect of our model is the use of heterologous training sets, in which peptide and non-peptide antimicrobial compounds were used to increase the size of the training data set. Our results show that combining peptide and non-peptide antimicrobial compounds rendered the best classification of gut antimicrobial compounds. Furthermore, this classification model was tested on the latest human-approved drugs expecting to identify antibiotics with broad-spectrum activity and our results show that the model rendered predictions consistent with current knowledge about broad-spectrum antibiotics. Therefore, heterologous machine learning rendered an efficient computational approach to classify antimicrobial compounds.
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Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Descoberta de Drogas , Aprendizado de Máquina , Bactérias/efeitos dos fármacos , Descoberta de Drogas/métodos , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Testes de Sensibilidade MicrobianaRESUMO
INTRODUCTION: Endothelial function at high altitude has been measured only in populations that are genetically adapted to chronic hypoxia. The objective of this study was to evaluate endothelial dysfunction (ED) in a nongenetically adapted high-altitude population of the Andes mountains, in Huancayo, Peru (3,250 meters above sea level). METHODS: Participants included 61 patients: 28 cases and 33 controls. The cases were subjects with hypertension, diabetes mellitus, obesity, or a history of stroke or coronary artery disease. Flow-mediated vasodilation (FMD) of the brachial artery was measured in the supine position, at noon, after 5 minutes of resting. The brachial artery was identified above the elbow. Its basal diameter was measured during diastole, and FMD was tested after 5 minutes of forearm ischemia. Intima-media complex in the right carotid artery was also determined. An increase in the artery's baseline diameter <10% indicated a positive test. Endothelium-independent vasodilation was evaluated with sublingual nitrate administration. The intima-media complex in the right carotid artery was also measured. RESULTS: 100% of diabetics had ED; ED was also found in 68.8% of obese individuals, 55% of hypertensive patients, and 46.5% of controls. Age, height, body mass index, and waist diameter were higher in the cases as compared with the controls. A total of 57.9% (n=11) of the cases and 45.2% (n=19) of the controls presented ED. Patients without ED had a mean increase in brachial artery diameter of 23.16%, while in those with ED it was only 3.84%. Individuals with diabetes or hypertension had a greater thickness of the carotid artery intima media layer (1.092 versus 0.664 cm) (p=0.037). A positive test for ED was associated with a greater basal diameter of the brachial artery (4.66±0.62 versus 4.23±0.59 cm) (p=0.02). A total of 7 patients presented paradoxical response, developing posthyperemia vasoconstriction. DISCUSSION: The proportion of ED was high among controls and among patients with risk factors. Controls showed better FMD profiles than subjects studied in Tibet and the Himalayas.
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Altitude , Artéria Braquial/fisiopatologia , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus/fisiopatologia , Endotélio Vascular/fisiopatologia , Obesidade/fisiopatologia , Vasoconstrição , Vasodilatação , Aclimatação , Administração Sublingual , Idoso , Artéria Braquial/efeitos dos fármacos , Doenças Cardiovasculares/diagnóstico por imagem , Doenças Cardiovasculares/epidemiologia , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitratos/administração & dosagem , Obesidade/diagnóstico , Obesidade/epidemiologia , Peru/epidemiologia , Fluxo Sanguíneo Regional , Fatores de Risco , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagemRESUMO
Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.
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Tirosina/análogos & derivados , Tirosina/metabolismo , Debaryomyces/genética , Debaryomyces/metabolismo , Tirosina/genética , Transcrição Gênica , Leveduras , Sequências Reguladoras de Ácido Nucleico , Regiões Promotoras Genéticas , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo Real , Osmorregulação , Extremófilos , Estresse Salino , Nitrogênio/metabolismoRESUMO
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
Ustilago maydis, a dimorphic fungus causing corn smut disease, serves as an excellent model to study different aspects of cell development. This study shows the influence of chitosan, oligochitosan and glycol chitosan on cell growth and physiology of U. maydis. These biological macromolecules affected the cell growth of U. maydis. In particular, it was found that chitosan completely inhibited U. maydis growth at 1mg/mL concentration. Microscopic studies revealed swellings on the surface of the cells treated with the polymers, and chitosan caused complete destruction of the membrane and formation of vesicles on the periphery of the cell. Oligochitosan and chitosan caused changes in oxygen consumption, K(+) efflux and H(+)-ATPase activity. Oligochitosan induced a faster consumption of oxygen in the cells, while glycol chitosan provoked slower oxygen consumption. It is noteworthy that chitosan completely inhibited the fungal respiratory activity. The strongest effects were exhibited by chitosan in all evaluated aspects. These findings showed high sensitivity of U. maydis to chitosan and provided evidence for antifungal effects of chitosan derivatives. To our knowledge, this is a first report showing that chitosan and its derivatives affect the cell morphology and physiological processes in U. maydis.
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Antifúngicos/farmacologia , Membrana Celular/efeitos dos fármacos , Quitina/análogos & derivados , Quitosana/farmacologia , Ustilago/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Quitina/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Transporte de Íons/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Oligossacarídeos , Consumo de Oxigênio/efeitos dos fármacos , Potássio/metabolismo , Relação Estrutura-Atividade , Ustilago/metabolismo , Ustilago/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.
Assuntos
Complexo I de Transporte de Elétrons/química , Transporte de Elétrons , Glicerolfosfato Desidrogenase/química , NADH Desidrogenase/química , Oxirredutases/química , Sequência de Aminoácidos , Respiração Celular/fisiologia , Debaryomyces/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/fisiologia , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMO
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
In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Glicólise , Metaboloma , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/agonistas , Actinas/antagonistas & inibidores , Actinas/química , Anticorpos Antifúngicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Cinética , Metaboloma/efeitos dos fármacos , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Faloidina/farmacologia , Polimerização/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Tiazolidinas/farmacologia , Trealose/farmacologia , Moduladores de Tubulina/farmacologia , ViscosidadeRESUMO
It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NOâ») in D. hansenii after growing in 2 M NaCl. Results showed the presence of NOâ» and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4.
Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , Proteínas Fúngicas/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomycetales/enzimologia , Cloreto de Sódio/metabolismo , Ativação Transcricional , Tirosina/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismoRESUMO
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.
Assuntos
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
During stress, many organisms accumulate compatible solutes. These solutes must be eliminated upon return to optimal conditions as they inhibit cell metabolism and growth. In contrast, enzyme interactions optimize metabolism through mechanisms such as channeling of substrates. It was decided to test the (compatible solute) trehalose-mediated inhibition of some yeast glycolytic pathway enzymes known to associate and whether inhibition is prevented when enzymes are allowed to associate. Trehalose inhibited the isolated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hexokinase (HXK), but not aldolase (ALD) nor phosphoglycerate kinase (PGK). When these enzymes were mixed in pairs, both GAPDH and HXK were protected by either ALD or PGK acquiring the inhibition behavior of the resistant enzyme. GAPDH was not protected by HXK, albumin or lactate dehydrogenase (LDH). Also, ALD did not protect glucose 6-phosphate dehydrogenase (G6PDH), suggesting that protection is specific. In yeast cell extracts, fermentation was resistant to trehalose inhibition, suggesting all enzymes involved in the glucose-dependent production of ethanol were stabilized. It is suggested that during the yeast stress response, enzyme association protects some metabolic pathways against trehalose-mediated inhibition.
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Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Hexoquinase/metabolismo , Fosfoglicerato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trealose/metabolismo , Inibidores Enzimáticos , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/antagonistas & inibidores , Glicólise , Hexoquinase/antagonistas & inibidores , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Fosfoglicerato Quinase/antagonistas & inibidores , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Trealose/farmacologiaRESUMO
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.