The familial amyotrophic lateral sclerosis-associated A4V SOD1 mutant is not able to regulate aerobic glycolysis.

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.

SOD1, more than just an antioxidant.

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.

Characterization of the activity, aggregation, and toxicity of heterodimers of WT and ALS-associated mutant Sod1.

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.

In Vivo Characterization of I91T Sod2 Polymorphism of Saccharomyces cerevisiae.

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.

Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain.

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.

Ischemia-reperfusion rat model of acute pancreatitis: protein carbonyl as a putative early biomarker of pancreatic injury.

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.

Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae

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.

Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae.

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.

Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose-xylose blend.

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.

Protective effects of flavonoids and extract from Vellozia kolbekii Alves against oxidative stress induced by hydrogen peroxide in yeast.

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.

New insights into the Ca2+-ATPases that contribute to cadmium tolerance in yeast.

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.

Evaluation of antimicrobial, antioxidant and phototoxic activities of extracts and isolated compounds from Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae / Avalição da atividade antimicrobiana, antioxidante e fototóxica dos extratos e compostos isolados de Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae

Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, plant extract, is a Brazilian medicinal plant externally used in folk medicine for purulent ulcers, skin lesions and internally for inflammations, fever, renal disorders and atherosclerosis. S. cayennesis was studied to identify potential bioactive compounds that may justify their therapeutic use against skin lesions and atherosclerosis. The antioxidant, antimicrobial and phototoxicity capacities of the crude ethanolic extract, fractions and isolated compounds from roots of S. cayennesis were evaluated through in vivo and in vitro tests. Strains of Saccharomyces cerevisiae, an eukaryotic cell model, were used to assess both the phototoxicity and the capacity to protect against the lethal oxidative stress caused by menadione and hydrogen peroxide. The extract, fractions and the two major isolated compounds, verbascoside and betulinic acid, of S. cayennensis were able to increase the tolerance and decrease the lipid peroxidation of S. cerevisiae to reactive oxygen species (ROS). The antioxidant activity was also evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH•). Verbascoside exhibited a moderate antimicrobial activity against Streptococcus pyogenes, S.epidermidis and Staphylococcus aureus. Neither the ethanolic extract nor fractions showed phototoxicity, indicating that the S. cayennensis extract is safe for use in the treatment of skin lesions and as an active cosmetic ingredient.

Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, é uma planta utilizada na medicina popular brasileira contra úlceras e lesões de pele. Internamente é usada contra inflamações, febre, doenças renais e aterosclerose. Essa planta foi estudada com o objetivo de identificar os compostos bioativos majoritários que possam justificar seu uso terapêutico contra lesões de pele e arteriosclerose. A atividade antioxidante do extrato bruto etanólico, partições e os compostos majoritários isolados das raízes de S. cayennesis foi avaliada através de testes in vivo e in vitro. In vitro a atividade antioxidante foi avaliada pelo teste fotocolorimétrico do radical 2,2-difenil-1-picrilidrazil (DPPH•). In vivo, Saccharomyces cerevisiae, um modelo de célula eucariótica, foi utilizado tanto para avaliar a fototoxicidade quanto a capacidade antioxidante contra as espécies reativas de oxigênio (EROS) menadiona e peróxido de hidrogênio. O extrato, partições e os dois compostos majoritários isolados, verbascosídeo e ácido betulínico foram capazes de aumentar a sobrevivência e diminuir a peroxidação lipídica de S. cerevisiae contra EROS. Verbascosídeo apresentou atividade antimicrobiana moderada contra Streptococcus pyogenes, S.epidermidis e Staphylococcus aureus. O extrato etanólico e as partições testadas não apresentaram fototoxicidade, indicando que S. cayennensis é uma planta segura para o tratamento de lesões de pele e como possível ingrediente em cosméticos.

Blood carbonyl protein measurement as a specific oxidative stress biomarker after intestinal reperfusion in rats.

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.

Blood carbonyl protein measurement as a specific oxidative stress biomarker after intestinal reperfusion in rats / Dosagem da proteína carbonilada sanguínea como biomarcador específico do estresse oxidativo após reperfusão intestinal em ratos

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.

OBJETIVO: Realizou-se um estudo experimental com a finalidade de investigar o uso da proteína carbonilada como um marcador biológico específico do estresse oxidativo em um modelo de isquemia e reperfusão intestinal, em ratos. MÉTODOS: Vinte e quarto ratos da linhagem Wistar, machos foram distribuídos, aleatoriamente, em três grupos compostos por oito animais cada: Grupo 1 - Controle; Grupo 2 - Simulação e Grupo 3 - Submetido à isquemia, mediante clampeamento de ramos ileais da artéria mesentérica superior por uma hora, seguida de reperfusão, por igual período. Amostras sanguíneas obtidas foram utilizadas para analise dos níveis de proteína carbonilada, através do método Slot blotting. RESULTADOS: No grupo 3 houve uma elevação significante da concentração de proteína carbonilada sérica se comparada aos níveis sanguíneos homogêneos encontrados nos grupos 1 e 2. CONCLUSÃO: Fundamentado nos resultados é possível concluir que, a carbonilação protéica pode ser utilizada como um marcador específico para a mensuração do estresse oxidativo em modelos de reperfusão intestinal, em ratos.

Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex.

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.

Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cerevisiae.

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.

Propofol and N-Acetylcysteine attenuate oxidative stress induced by intestinal ischemia/reperfusion in rats: Protein carbonyl detection by immunoblotting / Propofol e N-Acetilcisteína atenuam o estresse oxidativo induzido pela isquemia/reperfusão intestinal em ratos: Detecção de proteína carbonilada por immunoblotting

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.

OBJETIVO: Avaliar o efeito antioxidante do Propofol e N-Acetilcisteína (NAC) na isquemia/reperfusão (I/R) intestinal em ratos através da determinação do nível de proteína carbonilada. MÉTODOS: 40 ratos Wistar foram aleatoriamente distribuídos nos seguintes grupos: Controle; Sham; I/R com Propofol; I/R com Propofol e NAC; I/R com Ketamina e Xilazina. Os grupos I/R foram submetidos à isquemia durante 60 minutos e à reperfusão por igual período de tempo. Amostras de sangue, coletadas por punção cardíaca, foram centrifugadas para a obtenção de plasma. O nível de proteína carbonilada nas amostras de plasma foi determinado por imunoblotting. RESULTADOS: Nenhuma diferença significativa foi encontrada entre os grupos Controle e Sham (P>0.05). Uma redução marcante no nível de proteína carbonilada (P<0.05) foi obtida com a administração combinada de Propofol e NAC (Grupo 4) durante o procedimento de I/R intestinal, comparando-se com os demais grupos I/R testados. CONCLUSÃO: A administração de Propofol e NAC apresentou o melhor efeito antioxidante sobre o estresse oxidativo em ratos submetidos ao procedimento de I/R intestinal, sugerindo-se uma interação sinergística.

Propofol and N-Acetylcysteine attenuate oxidative stress induced by intestinal ischemia/reperfusion in rats: protein carbonyl detection by immunoblotting.

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.

The role of trehalose and its transporter in protection against reactive oxygen species.

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.

The role of the yeast ATP-binding cassette Ycf1p in glutathione and cadmium ion homeostasis during respiratory metabolism.

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.