The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical t... more The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical therapeutics of perfusion keep causing serious problems in the patient's clinical recovery. Then, they constitute a challenge to resolve. The objective of this work is to discuss the intracellular mechanisms that lead cells to apoptosis during the ischemia-reperfusion process, taking into consideration that these phenomena are observable in a simple microorganism as the yeast Saccharomyces cerevisiae. Yeast provide an alternative study system in which the effects of certain cytoprotective drugs can be evaluated. The results can then be extrapolated to other types of cells. Several works have focused on the role of mitochondria in the apoptotic processes of cellular necrosis. One of the main factors responsible for this process is the unregulated opening of the permeability barrier. The inner membrane thus allows the unrestricted passage of ions and the release of apoptotic mediators from the inner membrane space towards the cytosol. Also, there is an increase in the level of reactive oxygen species (ROS) and the uncoupling of oxidative phosphorylation, which lead to the reversal of ATP synthesis to ATP hydrolysis. The driving cause of this complex process is the opening of an non-specific pore located in the mitochondrial membrane, denominated mammalian permeability transition pore (mPTP), which is also expressed in yeast (yPTP). From the functional point of view, the yeast pore presents some of the characteristics observed in mammals, and is similar in the defensive response against the deleterious mechanisms caused by oxidative stress. An increasing body of evidence supports the concept that the pharmacological inhibition of the mPTP is an actual and promising strategy for the protection of tissues in ischemic situations in order to avoid the damage induced by perfusion.
In sepsis, reactive oxygen species (ROS) production is increased. This process takes place mainly... more In sepsis, reactive oxygen species (ROS) production is increased. This process takes place mainly within the electron transport chain. ROS production is part of the pathophysiology of multiple organ failure in sepsis. Succinate yields Dihydroflavine-Adenine Dinucleotide (FADH2), which enters the chain through complex II, avoiding complex I, through which electrons are lost. The aim of this work is to determine if parenteral succinate reduces systemic ROS production and improves kidney function. Rats with cecal ligation and puncture were used as model of sepsis, and 4 groups were made: Control group; Succinate group, which only received parenteral succinate; Sepsis group; and Sepsis which received parenteral succinate. Systemic ROS are measured 24 hours after the procedure. Rats subjected to cecal puncture treated with succinate had less systemic ROS than Septic untreated rats (p=0.007), while there were no differences in creatinine levels (p=0.07). There was no correlation between c...
Severe mycotic infections are a source of concern in immunocompromised patients or in those who r... more Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.
L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, ... more L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.
In this report, we present a practical approach to teaching several topics in nutrition to scienc... more In this report, we present a practical approach to teaching several topics in nutrition to science students at the high school and college freshmen levels. This approach uses baker's yeast (Saccharomyces cerevisiae) as a biological system model. The diameters of yeast colonies, which vary according to the nutrients present in the medium, can be observed, compared, and used to teach metabolic requirements. The experiments described in this report show simple macroscopic evidence of submicroscopic nutritional events. This can serve as a useful base for an analogy of heterotrophic human cell nutrition.
We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In... more We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.
Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the... more Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.
We investigated the damage caused by oxidative stress using the yeast Saccharomyces cerevisiae as... more We investigated the damage caused by oxidative stress using the yeast Saccharomyces cerevisiae as a model biological system. After inducing oxidative stress with menadione, we were able to evaluate the extent of cellular oxidative stress by utilizing 2',7'-dichlorofluorescein diacetate (DCFH-DA) as a marker of the presence of reactive oxygen species. Cells were grown on different carbon sources in order to compare fermentative and oxidative metabolism. Under these conditions we evaluated the effectiveness of idebenone (2,3-dimethoxy-5-methyl-6-(10- hydroxydecyl)-1,4-benzoquinone) as a molecule that could relieve menadione-induced growth inhibition in Saccharomyces cerevisiae.
The aim of this work was to study the effect of the drug idebenone on the growth of a strain of S... more The aim of this work was to study the effect of the drug idebenone on the growth of a strain of Saccharomyces cerevisiae yeast and its respiratory-deficient mutant (rho0). We took this yeast as a model system of the interaction of the drug with mammalian cells. The effect of idebenone was evaluated in rich and minimal media. In the S288c strain, idebenone exerted a growth inhibitory effect in concentrations higher than 50 μM in media containing a carbon source consumed at mitochondrial level. In conditions of low oxygen supply, idebenone allows yeast to keep a cellular yielding comparable with conditions of normal oxygen supply. Also, the presence of idebenone in the growth media increased by 50% the fluorescence signal of rhodamine 123, indicating a higher mitochondrial membrane potential. The results could explain the effect of idebenone in the treatment of diseases in which oxygen deficiency alters the energetic metabolism of the cell.
The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical t... more The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical therapeutics of perfusion keep causing serious problems in the patient's clinical recovery. Then, they constitute a challenge to resolve. The objective of this work is to discuss the intracellular mechanisms that lead cells to apoptosis during the ischemia-reperfusion process, taking into consideration that these phenomena are observable in a simple microorganism as the yeast Saccharomyces cerevisiae. Yeast provide an alternative study system in which the effects of certain cytoprotective drugs can be evaluated. The results can then be extrapolated to other types of cells. Several works have focused on the role of mitochondria in the apoptotic processes of cellular necrosis. One of the main factors responsible for this process is the unregulated opening of the permeability barrier. The inner membrane thus allows the unrestricted passage of ions and the release of apoptotic mediators from the inner membrane space towards the cytosol. Also, there is an increase in the level of reactive oxygen species (ROS) and the uncoupling of oxidative phosphorylation, which lead to the reversal of ATP synthesis to ATP hydrolysis. The driving cause of this complex process is the opening of an non-specific pore located in the mitochondrial membrane, denominated mammalian permeability transition pore (mPTP), which is also expressed in yeast (yPTP). From the functional point of view, the yeast pore presents some of the characteristics observed in mammals, and is similar in the defensive response against the deleterious mechanisms caused by oxidative stress. An increasing body of evidence supports the concept that the pharmacological inhibition of the mPTP is an actual and promising strategy for the protection of tissues in ischemic situations in order to avoid the damage induced by perfusion.
Carnitine is well-known for its role in the transport of fatty acids to the mitochondrial matrix,... more Carnitine is well-known for its role in the transport of fatty acids to the mitochondrial matrix, where beta-oxidation takes place. This work describes novel functions for this compound and novel data on its pharmacokinetics.
The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical t... more The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical therapeutics of perfusion keep causing serious problems in the patient's clinical recovery. Then, they constitute a challenge to resolve. The objective of this work is to discuss the intracellular mechanisms that lead cells to apoptosis during the ischemia-reperfusion process, taking into consideration that these phenomena are observable in a simple microorganism as the yeast Saccharomyces cerevisiae. Yeast provide an alternative study system in which the effects of certain cytoprotective drugs can be evaluated. The results can then be extrapolated to other types of cells. Several works have focused on the role of mitochondria in the apoptotic processes of cellular necrosis. One of the main factors responsible for this process is the unregulated opening of the permeability barrier. The inner membrane thus allows the unrestricted passage of ions and the release of apoptotic mediators from the inner membrane space towards the cytosol. Also, there is an increase in the level of reactive oxygen species (ROS) and the uncoupling of oxidative phosphorylation, which lead to the reversal of ATP synthesis to ATP hydrolysis. The driving cause of this complex process is the opening of an non-specific pore located in the mitochondrial membrane, denominated mammalian permeability transition pore (mPTP), which is also expressed in yeast (yPTP). From the functional point of view, the yeast pore presents some of the characteristics observed in mammals, and is similar in the defensive response against the deleterious mechanisms caused by oxidative stress. An increasing body of evidence supports the concept that the pharmacological inhibition of the mPTP is an actual and promising strategy for the protection of tissues in ischemic situations in order to avoid the damage induced by perfusion.
In sepsis, reactive oxygen species (ROS) production is increased. This process takes place mainly... more In sepsis, reactive oxygen species (ROS) production is increased. This process takes place mainly within the electron transport chain. ROS production is part of the pathophysiology of multiple organ failure in sepsis. Succinate yields Dihydroflavine-Adenine Dinucleotide (FADH2), which enters the chain through complex II, avoiding complex I, through which electrons are lost. The aim of this work is to determine if parenteral succinate reduces systemic ROS production and improves kidney function. Rats with cecal ligation and puncture were used as model of sepsis, and 4 groups were made: Control group; Succinate group, which only received parenteral succinate; Sepsis group; and Sepsis which received parenteral succinate. Systemic ROS are measured 24 hours after the procedure. Rats subjected to cecal puncture treated with succinate had less systemic ROS than Septic untreated rats (p=0.007), while there were no differences in creatinine levels (p=0.07). There was no correlation between c...
Severe mycotic infections are a source of concern in immunocompromised patients or in those who r... more Severe mycotic infections are a source of concern in immunocompromised patients or in those who receive chemotherapy for hematological malignant diseases. One of the causes is referred to the appearance of antimycotic resistant microorganisms. Fluconazole is one of the antimycotic used for invasive mycoses treatment. Therefore it is necessary to evaluate the factors that originate this resistance. In the present report the yeast Saccharomyces cerevisiae S288c was used as a model system. In resistant strains the accumulation of the lipophilic cation Rhodamine 6G, L-leucine uptake and growth inhibition by crystal violet dye were determined. The results presented herein demonstrate the correlation between the membrane potential and the resistance to fluconazole presented by S. cerevisiae strain S288c.
L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, ... more L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.
In this report, we present a practical approach to teaching several topics in nutrition to scienc... more In this report, we present a practical approach to teaching several topics in nutrition to science students at the high school and college freshmen levels. This approach uses baker's yeast (Saccharomyces cerevisiae) as a biological system model. The diameters of yeast colonies, which vary according to the nutrients present in the medium, can be observed, compared, and used to teach metabolic requirements. The experiments described in this report show simple macroscopic evidence of submicroscopic nutritional events. This can serve as a useful base for an analogy of heterotrophic human cell nutrition.
We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In... more We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.
Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the... more Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.
We investigated the damage caused by oxidative stress using the yeast Saccharomyces cerevisiae as... more We investigated the damage caused by oxidative stress using the yeast Saccharomyces cerevisiae as a model biological system. After inducing oxidative stress with menadione, we were able to evaluate the extent of cellular oxidative stress by utilizing 2',7'-dichlorofluorescein diacetate (DCFH-DA) as a marker of the presence of reactive oxygen species. Cells were grown on different carbon sources in order to compare fermentative and oxidative metabolism. Under these conditions we evaluated the effectiveness of idebenone (2,3-dimethoxy-5-methyl-6-(10- hydroxydecyl)-1,4-benzoquinone) as a molecule that could relieve menadione-induced growth inhibition in Saccharomyces cerevisiae.
The aim of this work was to study the effect of the drug idebenone on the growth of a strain of S... more The aim of this work was to study the effect of the drug idebenone on the growth of a strain of Saccharomyces cerevisiae yeast and its respiratory-deficient mutant (rho0). We took this yeast as a model system of the interaction of the drug with mammalian cells. The effect of idebenone was evaluated in rich and minimal media. In the S288c strain, idebenone exerted a growth inhibitory effect in concentrations higher than 50 μM in media containing a carbon source consumed at mitochondrial level. In conditions of low oxygen supply, idebenone allows yeast to keep a cellular yielding comparable with conditions of normal oxygen supply. Also, the presence of idebenone in the growth media increased by 50% the fluorescence signal of rhodamine 123, indicating a higher mitochondrial membrane potential. The results could explain the effect of idebenone in the treatment of diseases in which oxygen deficiency alters the energetic metabolism of the cell.
The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical t... more The apoptotic phenomena observed in tissues which are subdued to ischemia and then to technical therapeutics of perfusion keep causing serious problems in the patient's clinical recovery. Then, they constitute a challenge to resolve. The objective of this work is to discuss the intracellular mechanisms that lead cells to apoptosis during the ischemia-reperfusion process, taking into consideration that these phenomena are observable in a simple microorganism as the yeast Saccharomyces cerevisiae. Yeast provide an alternative study system in which the effects of certain cytoprotective drugs can be evaluated. The results can then be extrapolated to other types of cells. Several works have focused on the role of mitochondria in the apoptotic processes of cellular necrosis. One of the main factors responsible for this process is the unregulated opening of the permeability barrier. The inner membrane thus allows the unrestricted passage of ions and the release of apoptotic mediators from the inner membrane space towards the cytosol. Also, there is an increase in the level of reactive oxygen species (ROS) and the uncoupling of oxidative phosphorylation, which lead to the reversal of ATP synthesis to ATP hydrolysis. The driving cause of this complex process is the opening of an non-specific pore located in the mitochondrial membrane, denominated mammalian permeability transition pore (mPTP), which is also expressed in yeast (yPTP). From the functional point of view, the yeast pore presents some of the characteristics observed in mammals, and is similar in the defensive response against the deleterious mechanisms caused by oxidative stress. An increasing body of evidence supports the concept that the pharmacological inhibition of the mPTP is an actual and promising strategy for the protection of tissues in ischemic situations in order to avoid the damage induced by perfusion.
Carnitine is well-known for its role in the transport of fatty acids to the mitochondrial matrix,... more Carnitine is well-known for its role in the transport of fatty acids to the mitochondrial matrix, where beta-oxidation takes place. This work describes novel functions for this compound and novel data on its pharmacokinetics.
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