Alcoholic steatosis (AS) is the initial pathology associated with early stage alcoholic liver disease and is characterized by the accumulation of fat in the liver. AS is considered clinically benign as it is reversible, as compared with alcoholic steatohepatitis (ASH) which is the next stage of alcoholic liver disease (ALD), and mostly irreversible. Proteomics were used to investigate the molecular basis of AS to determine biomarkers representative of AS. Liver tissue proteins at different stages of steatosis from a rodent model of AS were separated by two dimensional electrophoresis (2DE), followed by MALDI mass spectrometry (MS) identification of significantly expressed proteins. Expression levels of several proteins related to alcohol induced oxidative stress, such as peroxiredoxin 6 (PRDX6) and aldehyde dehydrogenase 2 (ALDH2) were reduced by 2 to 3-fold in ethanol fed rats, and suggested an increase in oxidative stress. Several proteins involved in fatty acid and amino acid metabolism were found at increased expression levels, suggesting higher energy demand upon chronic exposure to ethanol. In order to delineate between the effects of fat accumulation and oxidative stress, an in vitro hepatocyte cell culture model of steatosis was developed. HepG2 cells loaded with oleic acid surprisingly demonstrated lower cytotoxicity upon oxidative challenge (based on lactate dehydrogenase activity) and inflammation (based on TNF-? induced activation of the pro-inflammatory transcription factor NF-?B). We also examined the effect of oleic acid loading in HepG2 cells on protein carbonylation, which is an important irreversible protein modification during oxidative stress that leads to protein dysfunction and disease. Fat-loaded hepatocytes exposed to oxidative stress with tert-butyl hydroperoxide (TBHP) contained 17% less carbonylated proteins than the non-fat loaded control. Mass spectrometric analysis of carbonylated proteins indicated that known classical markers of protein carbonylation (e.g., cytoskeletal proteins, chaperones) are not carbonylated in oleic acid loaded HepG2 cells, and suggests that the protective effect of fat loading is through interference with protein carbonylation. While counterintuitive to the general concept that AS increases oxidative stress, our fat loading results suggests that low levels of fat may activate antioxidant pathways and ameliorate the effect of subsequent oxidative or inflammatory challenge.

Alcoholic steatosis (AS) is the initial pathology associated with early stage alcoholic liver disease and is characterized by the accumulation of fat in the liver. AS is considered clinically benign as it is reversible, as compared with alcoholic steatohepatitis (ASH) which is the next stage of alcoholic liver disease (ALD), and mostly irreversible. Proteomics were used to investigate the molecular basis of AS to determine biomarkers representative of AS. Liver tissue proteins at different stages of steatosis from a rodent model of AS were separated by two dimensional electrophoresis (2DE), followed by MALDI mass spectrometry (MS) identification of significantly expressed proteins. Expression levels of several proteins related to alcohol induced oxidative stress, such as peroxiredoxin 6 (PRDX6) and aldehyde dehydrogenase 2 (ALDH2) were reduced by 2 to 3-fold in ethanol fed rats, and suggested an increase in oxidative stress. Several proteins involved in fatty acid and amino acid metabolism were found at increased expression levels, suggesting higher energy demand upon chronic exposure to ethanol. In order to delineate between the effects of fat accumulation and oxidative stress, an in vitro hepatocyte cell culture model of steatosis was developed. HepG2 cells loaded with oleic acid surprisingly demonstrated lower cytotoxicity upon oxidative challenge (based on lactate dehydrogenase activity) and inflammation (based on TNF-? induced activation of the pro-inflammatory transcription factor NF-?B). We also examined the effect of oleic acid loading in HepG2 cells on protein carbonylation, which is an important irreversible protein modification during oxidative stress that leads to protein dysfunction and disease. Fat-loaded hepatocytes exposed to oxidative stress with tert-butyl hydroperoxide (TBHP) contained 17% less carbonylated proteins than the non-fat loaded control. Mass spectrometric analysis of carbonylated proteins indicated that known classical markers of protein carbonylation (e.g., cytoskeletal proteins, chaperones) are not carbonylated in oleic acid loaded HepG2 cells, and suggests that the protective effect of fat loading is through interference with protein carbonylation. While counterintuitive to the general concept that AS increases oxidative stress, our fat loading results suggests that low levels of fat may activate antioxidant pathways and ameliorate the effect of subsequent oxidative or inflammatory challenge.

No description available.

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.

La photocatalyse est un procédé d'oxydation avancée qui consiste en l'activation du dioxyde de titane sous UV pour générer des espèces oxydantes. Ces dernières sont capables d'inactiver les cellules vivantes. Nos travaux ont porté sur l'analyse des mécanismes antimicrobiens de la photocatalyse à l'échelle cellulaire et moléculaire sur le modèle eucaryote Saccharomyces cerevisiae, champignon unicellulaire. Le traitement photocatalytique affecte de manière drastique la cultivabilité de cette levure. La diminution de la cultivabilité a été reliée à la perte de l'intégrité membranaire et à la perte de l'activité enzymatique intracellulaire, analysées par cytométrie en flux. L'exposition des levures à la photocatalyse provoque des dommages à toutes les macromolécules (acides nucléiques, lipides membranaires, protéines) et par conséquent aux structures cellulaires ce qui engendre la libération de constituants cellulaires (ions, acides aminés), de même que la formation de produits de dégradation (malondialdéhyde, acides organiques). Ces dommages peuvent être liés à un stress oxydant intracellulaire suggéré par l'accumulation des ions superoxyde dans les cellules traitées et l'augmentation de la résistance pour les souches surexprimant des enzymes de dégradation des ROS. Enfin, l'étude de l'impact de la photocatalyse sur des organismes fongiques ayant un impact environnemental ou sur la santé, a révélé l'existence de cellules ou de structures fongiques résistantes. Ces résultats ont apporté des éléments de connaissance inédits sur l'impact de la photocatalyse sur les cellules eucaryotes fongiques et ouvrent de nouvelles perspectives notamment dans la compréhension du phénomène de résistance / Photocatalysis is an advanced oxidative process that generates reactive oxygen species (ROS) and inactivates living cells. The aim of this work was to have a better understanding of the antimicrobial mechanisms generated by photocatalytic treatment. The cellular impact was monitored using the unicellular fungal model, Saccharomyces cerevisiae yeast. Photocatalysis reduces drastically the cultivability of yeast cells. Flow cytometry analyses revealed that the decrease of cell cultivability was related to both damages in plasma membrane and loss of intracellular enzymatic activity. During exposure to photocatalysis, multiple cellular macromolecules are damaged (lipids, proteins, nucleic acids). These damages are responsible for cellular structure dysfunction leading to a release of intracellular compounds (ions, amino acids) and the formation of by-products and pollutant (carboxylic acids, malondialdéhyde). The increase of intracellular superoxide ions amounts and the higher resistance of yeast strains overexpressing ROS detoxifying enzymes suggested an intracellular oxidative status responsible for described macromolecular damages. Finally, exploring photocatalytic treatment on other environmental and health impact fungi revealed the presence of resistant cells or structures. For the first time, an interdisciplinary work focusing on cellular impacts of photocatalysis was monitored leading to a better understanding and to new perspectives

Traumatic Brain Injury (TBI) is the major cause of death or cognitive deficits in industrialized countries. Although studies have indicate that the oxidative stress and functional deficits after TBI are connected events, the mechanisms that outline the development of these cognitive deficits are, still, limited. In this context, we investigated the involvement of oxidative stress markers (thiobarbituric acid reactive species; TBARS and protein carbonylation) and the Na+,K+-ATPase enzyme activity on the spatial learning after one and three months from a fluid percussion injury (FPI) in rats. The results revealed that FPI increase the latency of escape and the number of the errors on the Barnes Maze Test one and three months after FPI. We also found an increase of TBARS and protein carbonylation in parietal cortex after one and three months FPI. In addition, statistical analysis revealed a decrease of the Na+,K+- ATPase enzyme activity in the parietal cortex after FPI (time-dependent). These results suggest that cognitive impairment following FPI may result, at least in part, from increase of two oxidative stress markers, protein carbonylation and TBARS that occurs concomitantly to a decrease in Na+,K+-ATPase activity. Physical exercise, despite the involvement on the generation of the reactive oxygen species (ROS), is used on the rehabilitation of TBI. However, although the favorable effects of physical exercise on traumatic brain injury (TBI) patients is well known, the specific mechanisms involved in this protection after TBI has been limited. Thus, we investigated whether physical training protects against oxidative damage (measured by protein carbonylation and TBARS) and neurochemical alterations represented by immunodetection of alpha subunit and activity of Na+,K+-ATPase after FPI in cerebral cortex of rats. The results revealed that physical training protected against oxidative damage induced by FPI. In addition, physical training was effective against Na+,K+- ATPase enzyme activity inhibition and α subunit level decrease after FPI. The Pearson correlation showed that the decrease of the catalytical levels of the Na+,K+- ATPase enzyme α subunit is related with the increasing on oxidative stress markers. Moreover, the physical activity-related protection against free radicals induced by FPI links with maintenance of α subunit immunocontent. These results suggest that the effective protection stimulated by physical exercise on the neuronal damage induced by TBI has connection with the protection of the specific targets from the free radicals action, like Na+,K+-ATPase enzyme. / O Traumatismo crânio-encefálico (TCE) é uma das maiores causas de morte ou déficits cognitivos nos países industrializados. Apesar de os estudos indicarem que o estresse oxidativo e os déficits funcionais que ocorrem após TCE serem eventos interrelacionados, os mecanismos que delineiam o desenvolvimento destes déficits cognitivos são, ainda, limitados. Neste contexto nós investigamos o envolvimento de marcadores de estresse oxidativo (espécies reativas ao ácido tiobarbitúrico; TBARS e carbonilação protéica) e a atividade da enzima Na+,K+-ATPase no aprendizado espacial um e três meses após um dano de percussão por fluído (FPI) em ratos. Os resultados revelaram que o FPI aumentou o tempo de latência e o número de erros no teste do labirinto de Barnes em um e três meses após FPI. Também encontramos aumento no conteúdo de TBARS e proteína carbonil no córtex parietal em um e três meses após FPI. Além disso, a análise estatística revelou uma diminuição na atividade da enzima Na+,K+-ATPase no córtex cerebral após FPI tempo dependente, sugerindo que o déficit cognitivo induzido pelo FPI se deva pela perda de funcionabilidade de enzimas presentes na células como Na+,K+-ATPase. Perda esta induzida pelo aumento na geração de radicais livres após TCE. Apesar de estar envolvido no aumento da produção de espécies reativas ao Oxigênio (ERO), exercício físico tem sido utilizado na reabilitação de após TCE. Por outro lado, ainda são escassos na literatura estudos que evidenciam a especificidade dos mecanismos envolvidos na proteção induzida pelo exercício físico após TCE. Desta forma, investigamos se o treinamento físico protege contra o dano oxidativo bem como das alterações neuroquímicas representadas pela imunodetecção da subunidade α e da atividade da enzima Na+,K+-ATPase no córtex cerebral de ratos. Os resultados revelaram que o treinamento físico protegeu contra o dano oxidativo induzido por FPI. Além disso, o treinamento físico foi efetivo contra a inibição da enzima Na+,K+-ATPase e a diminuição dos níveis da sua subunidade α após FPI. A correlação de Pearson revelou que a diminuição dos níveis catalíticos da subunidade α da enzima Na+,K+-ATPase se correlaciona com o aumento dos marcadores de estresse oxidativo. Além disso, a proteção exercida pela atividade física contra os radicais livres induzidos por FPI tem relação com a manutenção do imunoconteúdo da subunidade α. A partir destes achados, sugere-se que a efetiva proteção exercida pelo exercício físico no dano neuronal causado induzido pelo TCE se deva pela proteção de alvos específicos a ação de radicais livres, como a enzima Na+, K+-ATPase.

Monosialoganglioside (GM1) is a component of most cell membranes and is thought to play a role in development, recognition and cellular differentiation. Furthermore, GM1 is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. In the present study we investigate the effect of GM1 on the catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, on the spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cortex of rats ex vivo and in vitro. Systemic GM1 administration (50 mg/kg, i.p.; twice) reduced spontaneous chemiluminescence and increased CAT activity ex vivo. On the other hand, GM1 (103-104 nM) reduced CAT activity in vitro. The other parameters were not affected by GM1 administration. These findings agree with the view that the antioxidant action of GM1 is not due to an intrinsic antioxidant activity of this glycolipid, but due to a secondary decrease of reactive species generation and/or increase of antioxidant defenses. Moreover, we evaluated whether GM1 could have a neuroprotective action on the experimental model of glutaric acidemia, an inherited metabolic disorder characterized by glutaric acid (GA) accumulation and neurological dysfunction, as striatal degeneration and convulsion. The systemic GM1 administration (50 mg/kg, i.p. twice) protected against the convulsions, oxidative damage markers increase (total protein carbonylation and thiobarbituric acid-reactive substances - TBARS) production and Na+,K+-ATPase activity inhibition induced by GA (4 mol/ 2 l) in striatum of rats. Furthermore, convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum, but not with oxidative stress marker measures. In addition, GM1 (50-200 M) protected against Na+,K+-ATPase inhibition induced by GA (6 mM), but not against oxidative damage in vitro. Intrastriatal administration of muscimol (46 pmol/striatum), a GABAA receptor agonist, but not glutamatergic receptor antagonists MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum), prevented GA-induced convulsions and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance in the injected striatum with GA. Since GM1 and muscimol prevented neurotoxic effects induced by GA, we investigated the GM1 action after intrastriatal administration of pentylenetetrazole (PTZ), a GABAA receptor antagonist. GM1 treatment prevented seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl induced by PTZ (1.8 mol/striatum) in the rats striatum. Furthermore, these data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1. / O monossialogangliosídeo (GM1) é um componente natural de membrana plasmática que está envolvido no crescimento, reconhecimento e diferenciação celular, além de proteger o SNC da ação dos radicais livres. No presente estudo investigou-se o efeito do GM1 sobre a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD) e glutationa peroxidase (GPx), assim como na quimiluminescência e capacidade antioxidante total (TRAP) em córtex cerebral de ratos machos adultos ex vivo e in vitro. A administração sistêmica de GM1 (50 mg/kg, i.p.; duas doses: 24 horas e 30 minutos antes do sacrifício) reduziu a quimiluminescência e aumentou significativamente a atividade da CAT ex vivo. A adição de GM1 (103-104 nM) ao meio de incubação diminuiu a atividade da CAT in vitro. Estes resultados sugerem que o efeito neuroprotetor do GM1 não é devido à ação antioxidante intrínseca deste glicoesfingolipídeo, mas devido ao aumento secundário das defesas antioxidantes e/ou uma redução da geração de radicais livres. Além disso, avaliamos se o GM1 tinha efeito neuroprotetor em um modelo experimental da acidemia glutárica, um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido glutárico (GA) e alterações neurológicas, como degeneração estriatal e convulsões. A administração de GM1 preveniu as convulsões, o aumento da produção dos marcadores do dano oxidativo (carbonilação protéica total e substâncias reativas do ácido tiobarbitúrico - TBARS) e a inibição da atividade da Na+,K+-ATPase induzidas pelo GA (4 mol/2 µl) em estriado de ratos. Além disso, os episódios convulsivos induzidos por GA apresentaram uma correlação significativa com a inibição da atividade da Na+,K+-ATPase no estriado injetado, mas não com os níveis dos marcadores do estresse oxidativo. A adição de GM1 (50 200  ao meio de incubação preveniu a inibição da Na+,K+-ATPase, mas não reduziu o dano oxidativo induzido por GA (6 mM) in vitro. A administração intraestriatal de muscimol (46 pmol/0,5 l), um agonista de receptor GABAA, mas não dos antagonistas de receptores glutamatérgicos, MK-801 (3 nmol/0,5 l) e DNQX (8 nmol/0,5 l), preveniu as convulsões e a inibição da atividade da Na+,K+-ATPase induzidas por GA. A proteção do GM1 e muscimol contra as convulsões induzidas por GA apresentou uma correlação significativa com a manutenção da atividade da Na+,K+-ATPase no estriado injetado com GA. Desde que o GM1 e o muscimol preveniram os efeitos neurotóxicos induzidos pelo GA, investigou-se a ação do GM1 após a administração intraestriatal de pentilenotetrazol (PTZ), um antagonista de receptores GABAA. O tratamento com GM1 preveniu as convulsões, o dano oxidativo e a inibição da atividade da Na+,K+-ATPase induzidas por PTZ (1,8 µmol/2 µl). Esses dados sugerem que a atividade da Na+,K+-ATPase e mecanismos mediados pela ativação de receptores GABAérgicos podem ser de grande importância para a atividade convulsiva induzida por GA, bem como nos mecanismos de neuroproteção induzidos pelo GM1.