RESUMO
Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Envelhecimento , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Corpos de Inclusão/metabolismo , Peso Molecular , Proteínas Mutantes/química , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/químicaRESUMO
During cellular respiration, radicals, such as superoxide, are produced, and in a large concentration, they may cause cell damage. To combat this threat, the cell employs the enzyme Cu/Zn Superoxide Dismutase (SOD1), which converts the radical superoxide into molecular oxygen and hydrogen peroxide, through redox reactions. Although this is its main function, recent studies have shown that the SOD1 has other functions that deviates from its original one including activation of nuclear gene transcription or as an RNA binding protein. This comprehensive review looks at the most important aspects of human SOD1 (hSOD1), including the structure, properties, and characteristics as well as transcriptional and post-translational modifications (PTM) that the enzyme can receive and their effects, and its many functions. We also discuss the strategies currently used to analyze it to better understand its participation in diseases linked to hSOD1 including Amyotrophic Lateral Sclerosis (ALS), cancer, and Parkinson.
Assuntos
Antioxidantes/metabolismo , Superóxido Dismutase-1/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/química , Saúde , Humanos , Superóxido Dismutase-1/químicaRESUMO
The mitochondrial antioxidant enzyme Mn-Superoxide Dismutase (Sod2) is essential for mammalian survival. I82T mutation in human Sod2 has been linked to a wide variety of diseases, including Alzheimer's and Parkinson's diseases as well as some types of cancers. Yeast wild-type (WT) Sod2 and the mutant Sod2 I91T, which corresponds to the human mutant Sod2 I82T, were cloned in sod2Δ strain. Residue I82 is conserved among a variety of species, showing that it has a biological importance. To assess the functionality of Sod2 I91T under oxidative stress, yeast cells were shifted from glucose (fermentative metabolism) to glycerol growth medium (respiratory metabolism). Overexpression of both Sod2 WT and Sod2 I91T increased Sod activity, but in long-term, the mutation brought impairment to Sod function. Aconitase, a sensor of superoxide radical production in vivo, had its activity preserved by overexpressions of both Sod2, in lesser extent in sod2ΔSod2I91T. In respiratory metabolism, sod2ΔSod2WT and sod2ΔSod2I91T showed high viability; although, sod2ΔSod2I91T showed high percentage of cells with mitochondrial function compromised. Moreover, the fitness analysis of mixed cultures showed that sod2ΔSod2I91T was less robust than WT cells. Although overexpression of Sod2 containing I91T mutation allows higher cell viability, longevity of cells is hampered, showing that in long-term this mutation is not neutral. J. Cell. Biochem. 118: 1078-1086, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Glicerol/metabolismo , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/crescimento & desenvolvimento , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sequência de Bases , Sequência Conservada , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/fisiologia , Modelos Biológicos , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para CimaRESUMO
Under certain stress conditions, astrocytes operate in aerobic glycolysis, a process controlled by pyruvate dehydrogenase (PDH) inhibition through its E1 α subunit (Pda1) phosphorylation. This supplies lactate to neurons, which save glucose to obtain NADPH to, among other roles, counteract reactive oxygen species. A failure in this metabolic cooperation causes severe damage to neurons. In this work, using humanized Saccharomyces cerevisiae cells in which its endogenous Cu/Zn Superoxide Dismutase (SOD1) was replaced by human ortholog, we investigated the role of human SOD1 (hSOD1) in aerobic glycolysis regulation and its implications to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Yeast cells ferment glucose even in the presence of oxygen and switch to respiratory metabolism after glucose exhaustion. However, like cells of SOD1-knockout strain, cells expressing A4V mutant of hSOD1 growing on glucose showed a respiratory phenotype, i.e., low glucose and high oxygen consumptions and low intracellular oxidation levels in response to peroxide stress, contrary to cells expressing wild-type (WT) SOD1 (yeast or human). The A4V mutation in hSOD1 is linked to ALS. In contrast to WT SOD1 strains, PDH activity of both sod1Δ and A4V hSOD1 cells did not change in response to a metabolic shift toward oxidative metabolism, which was associated to lower Pda1 phosphorylation levels under growth on glucose. Taken together, our results suggest that A4V mutant cannot regulate aerobic glycolysis via Pda1 phosphorylation the same way WT hSOD1, which might be linked to problems observed in the motor neurons of ALS patients with the SOD1 A4V mutation.
Assuntos
Esclerose Lateral Amiotrófica , Glicólise , Saccharomyces cerevisiae , Superóxido Dismutase-1 , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , MutaçãoRESUMO
Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 muM CdSO(4), showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.
Assuntos
Apoptose/genética , Compostos de Cádmio/toxicidade , Glutarredoxinas/fisiologia , Mutagênicos/toxicidade , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Sulfatos/toxicidade , Glutarredoxinas/genética , Glutationa/metabolismo , Oxirredução , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
During menadione stress, trehalose was necessary intracellularly, but under H2O2, the sugar was required on the outside of the plasma membrane. The mechanism of protection involves minimizing the oxidative damage caused to both proteins and lipids, which would require the presence of trehalose on both sides of the lipid bilayer.
Assuntos
Proteínas de Transporte de Monossacarídeos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Simportadores/fisiologia , Trealose/fisiologia , Glucosiltransferases/genética , Peróxido de Hidrogênio/farmacologia , Peroxidação de Lipídeos , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Oxidantes/farmacologia , Estresse Oxidativo , Carbonilação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Simportadores/genética , Trealose/farmacologia , Vitamina K 3/farmacologiaRESUMO
Cadmium (Cd2+) is a toxic environmental contaminant for biological systems, which can form complexes with reduced glutathione (GSH), and thus alter the intracellular redox state. In Saccharomyces (S.) cerevisiae, bis(glutathionato)cadmium (Cd-[GS]2) complexes can be removed from the cytosol and transported into the vacuole by a glutathione-conjugated pump, Ycf1p. In this study, we investigated the role of Ycf1p in Cd2+ detoxification during respiratory metabolism of S. cerevisiae, and the correlation of Ycf1p with GSH intracellular homeostasis. The results showed that in respiratory condition the mutant ycf1Delta is more tolerant to Cd2+ and to the oxidants t-BOOH and H2O2 than wild-type strain. This tolerance is probably related to the high content of GSH present in ycf1Delta mutant. The expression of YCF1 promoter in the wild-type strain is naturally down-regulated after the transition from fermentative to respiratory metabolism (diauxic shift), and its induction in response to Cd2+ is dependent on GSH availability. Our data suggest that Ycf1p is involved in the maintenance of intracellular GSH homeostasis and it can interfere with the oxidative tolerance of yeast. Moreover, the detoxification of Cd2+ is dependent on GSH availability and on cellular metabolic status.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Cloreto de Cádmio/metabolismo , Glutationa/metabolismo , Homeostase/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Íons , Oxidantes/toxicidade , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , terc-Butil Hidroperóxido/toxicidadeRESUMO
In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.
Assuntos
Catalase/metabolismo , Saccharomyces cerevisiae/metabolismo , Catalase/genética , Glutationa/metabolismo , Peroxidação de Lipídeos , Mutação , Oxirredução , Estresse Oxidativo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose. The resulting yeast strain co-fermented glucose and xylose rapidly and almost simultaneously, exhibiting improved ethanol production and productivity. It was also observed that when cells were grown in a medium containing higher glucose concentrations before being transferred to fermentation medium, higher rates of xylose consumption and ethanol production were obtained, demonstrating that xylose utilization was not regulated by catabolic repression. Results obtained by qPCR demonstrate that the efficiency in xylose fermentation showed by the evolved strain is associated, to the increase in the expression of genes HXT2 and TAL1, which code for a low-affinity hexose transporter and transaldolase, respectively. The ethanol productivity obtained after the introduction of only one genetic modification and the submission to a one-stage process of evolutionary engineering was equivalent to those of strains submitted to extensive metabolic and evolutionary engineering, providing solid basis for future applications of this strategy in industrial strains.
RESUMO
Acute pancreatitis (AP) is an inflammatory disorder that can affect adjacent and/or remote organs. Some evidence indicates that the production of reactive oxygen species is able to induce AP. Protein carbonyl (PC) derivatives, which can also be generated through oxidative cleavage mechanisms, have been implicated in several diseases, but there is little or no information on this biomarker in AP. We investigated the association between some inflammatory mediators and PC, with the severity of ischemia-reperfusion AP. Wistar rats (n = 56) were randomly assigned in the following groups : control; sham, 15- or 180-min clamping of splenic artery, with 24 or 72 h of follow-up. The relationships between serum level of PC and thiobarbituric acid reactive species (TBARS) to myeloperoxidase (MPO) activity in tissue homogenates and to cytokines in culture supernatants of pancreatic samples were analyzed. MPO activity was related to the histology scores and increased in all clamping groups. Tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin-6 were higher in the 180-min groups. Significant correlations were found between MPO activity and the concentrations of TNF-α and IL-1ß. PC levels increased in the 15-min to 24-h group. TBARS levels were not altered substantially. MPO activity and TNF-α and IL-1ß concentrations in pancreatic tissue are correlated with AP severity. Serum levels of PC appear to begin to rise early in the course of the ischemia-reperfusion AP and are no longer detected at later stages in the absence of severe pancreatitis. These data suggest that PC can be an efficient tool for the diagnosis of early stages of AP.
Assuntos
Biomarcadores/análise , Pancreatite Necrosante Aguda/diagnóstico , Pancreatite Necrosante Aguda/patologia , Carbonilação Proteica , Traumatismo por Reperfusão/patologia , Animais , Citocinas/análise , Modelos Animais de Doenças , Feminino , Peroxidase/análise , Ratos WistarRESUMO
Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined. Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration. In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod. To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2',7'-dichlorofluorescein, was used. Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration. Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation.
Assuntos
Desidratação/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/enzimologia , Superóxido Dismutase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Superóxido Dismutase/genética , Trealose/metabolismoRESUMO
Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 °C, in 0.04% sulphite, 1 mol L(-1) NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors of substrates into ethanol (Y(p/s)), glycerol (Y(g/s)) and acetic acid (Y(ac/s)), were calculated. The highest values of Y(p/s) in sugar cane juice fermentation were obtained by four strains, one isolated from fruit (0.46) and the others from sugar cane (0.45, 0.44 and 0.43). These values were higher than the value obtained using traditional yeast (0.38) currently employed in the Brazilian bioethanol industry. The parameters Y(g/s) and Y(ac/s) were low for all strains. The UFLA FW221 presented the higher values for parameter related to bioethanol production. Thus, it was tested in co-culture with Lactobacillus fermentum. Besides this, a 20-L vessel for five consecutive batches of fermentation was performed. This strain was genetically stable and remained viable during all batches, producing high amounts of ethanol. The UFLA FW221 isolated from fruit was suitable to produce bioethanol in sugar cane juice. Therefore, the study of the biodiversity of yeasts from different environmental can reveal strains with desired characteristics to industrial applications.
Assuntos
Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Ácido Acético/metabolismo , Brasil , Metabolismo dos Carboidratos , Agregação Celular , Etanol/metabolismo , Etanol/toxicidade , Fermentação , Glicerol/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/efeitos da radiação , Cloreto de Sódio/metabolismo , Cloreto de Sódio/toxicidade , Sulfitos/metabolismo , Sulfitos/toxicidade , TemperaturaRESUMO
This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose-xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Burkholderia cenocepacia/fisiologia , Etanol/metabolismo , Glucose/metabolismo , Saccharomyces cerevisiae/fisiologia , Xilose/metabolismo , Aldose-Cetose Isomerases/genética , Etanol/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismoRESUMO
Two flavonoids 3,5,7,3',4'-pentahydroxy-6-prenylflavonol (1) and 3,5,7,3',4'-pentahydroxy-8-methyl-6-prenylflavonol (2) were isolated from the ethyl acetate extract of sheaths of Vellozia kolbekii Alves (Velloziaceae). This is the first time that compound 2 has been described. The crude extract and flavonoids were found to be active as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavengers and were able to the increase tolerance of the eukaryotic microorganism Saccharomyces cerevisiae to oxidative stress generated by H(2)O(2). The protective effect was correlated with a reduction in the oxidation of proteins and lipids. In addition, flavonoids isolated from Velloziaceae showed an inhibitory effect on mutations in p53, which is mutated and nonfunctional in more than 50% of cases of human cancer.
Assuntos
Flavonoides/farmacologia , Peróxido de Hidrogênio/toxicidade , Magnoliopsida/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Antioxidantes/química , Antioxidantes/farmacologia , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Extratos Vegetais/química , Carbonilação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismoRESUMO
Cadmium (Cd(2+)) is a toxic heavy metal which triggers several toxic effects in eukaryotes, including neurotoxicity and impaired calcium metabolism. In the model organism Saccharomyces cerevisiae, the best characterized pathway for Cd(2+) detoxification involves conjugation with glutathione (GSH) and subsequent transport to vacuoles by Ycf1p, an ATPase homologous to human MRP1 (Multidrug resistance associated protein 1). However, Cd(2+) tolerance also can be mediated by Pmr1p, a Ca(2+) pump located in the Golgi membrane, possibly through to the secretory pathway. Herein, we showed that inactivation of the PMR1 gene, alone or simultaneously with YCF1, delayed initial Cd(2+) capture compared to wild-type (WT) cells. In addition, Cd(2+) treatment altered the expression profile of yeast internal Ca(2+) transporters; specifically, PMC1 gene expression is induced substantially by the metal in WT cells, and this induction is stronger in mutants lacking YCF1. Taken together, these results indicate that, in addition to Pmr1p, the vacuolar Ca(2+)-ATPase Pmc1p also helps yeast cells cope with Cd(2+) toxicity. We propose a model where Pmc1p and Pmr1p Ca(2+)-ATPase function in cooperation with Ycf1p to promote Cd(2+) detoxification.
Assuntos
Cádmio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , Glutationa/metabolismo , Complexo de Golgi/metabolismo , Chaperonas Moleculares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/metabolismoRESUMO
PURPOSE: An experimental study was performed to investigate the use of protein carbonyl group as a specific biological marker for oxidative stress in a rat model of intestinal ischaemia-reperfusion. METHODS: Twenty four male Wistar rats were randomly distributed into three groups with eight animals each: Group 1 - Control group; Group 2 - Sham; Group 3 - Intestinal ischaemia by clamping ileal branches of the superior mesenteric artery for one hour, followed by another hour of reperfusion. Blood samples were taken in order to analyze the protein carbonyl level by Slot blotting assay. RESULTS: In group 3 a significant increase of protein carbonyl level was observed if compared to the homogenous levels of groups 1 and 2. CONCLUSION: From the results it may be concluded that the protein carbonylation may be used as a specific marker for measuring oxidative stress in rat intestinal reperfusion model.
Assuntos
Proteínas Sanguíneas/análise , Intestino Delgado/irrigação sanguínea , Estresse Oxidativo , Traumatismo por Reperfusão/sangue , Animais , Biomarcadores/análise , Modelos Animais de Doenças , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Traumatismo por Reperfusão/diagnósticoRESUMO
In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione.
Assuntos
Cádmio/metabolismo , Glutationa Transferase/metabolismo , Glutationa/metabolismo , gama-Glutamiltransferase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
BACKGROUND: Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. METHODOLOGY/PRINCIPAL FINDINGS: Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Delta and gtt2Delta) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. CONCLUSIONS/SIGNIFICANCE: These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Naftoquinonas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Vitamina K 3/farmacologia , Vitaminas/farmacologia , Glutationa/genética , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos , Testes de Sensibilidade Microbiana , Oxirredução , Estresse OxidativoRESUMO
PURPOSE: To evaluate the antioxidant effect of Propofol and N-Acetylcysteine (NAC) on intestinal ischemia/reperfusion (I/R) in rats by determining carbonyl protein level. METHODS: Forty Wistar rats were randomly assigned into the following groups: Control; Sham; I/R with Propofol; I/R with Propofol and NAC; I/R with Ketamine and Xylazine. The I/R groups underwent 60 minutes of ischemia and an equal period of reperfusion. Blood samples, collected by cardiac punction, were centrifuged for plasma obtainment. Protein carbonyl level in plasma samples was determined by immunoblotting. RESULTS: No significant difference in protein carbonyl level was found between Control and Sham groups (P>0.05). The highest reduction in protein carbonyl level (P<0.05) was obtained with the administration of Propofol and NAC (Group 4) in intestinal I/R procedure. CONCLUSION: The administration of Propofol and NAC showed the best antioxidant effect on oxidative stress in rats that underwent intestinal I/R procedure, suggesting a synergistic interaction.
Assuntos
Acetilcisteína/farmacologia , Sequestradores de Radicais Livres/farmacologia , Intestinos/irrigação sanguínea , Estresse Oxidativo/efeitos dos fármacos , Propofol/farmacologia , Carbonilação Proteica/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Biomarcadores/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/fisiopatologiaRESUMO
Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 ºC, in 0.04% sulphite, 1 mol L-1 NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors of substrates into ethanol (Yp/s), glycerol (Yg/s) and acetic acid (Yac/s), were calculated. The highest values of Yp/s in sugar cane juice fermentation were obtained by four strains, one isolated from fruit (0.46) and the others from sugar cane (0.45, 0.44 and 0.43). These values were higher than the value obtained using traditional yeast (0.38) currently employed in the Brazilian bioethanol industry. The parameters Yg/s and Yac/s were low for all strains. The UFLA FW221 presented the higher values for parameter related to bioethanol production. Thus, it was tested in co-culture with Lactobacillus fermentum. Besides this, a 20-L vessel for five consecutive batches of fermentation was performed. This strain was genetically stable and remained viable during all batches, producing high amounts of ethanol. The UFLA FW221 isolated from fruit was suitable to produce bioethanol in sugar cane juice. Therefore, the study of the biodiversity of yeasts from different environmental can reveal strains with desired characteristics to industrial applications.