The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Genetic dysregulation of glutathione synthesis predicts alteration of plasma thiol redox status in schizophrenia.

Abstract Genetic studies have shown an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL), the key enzyme for glutathione (GSH) synthesis. The present study was aimed at analyzing the influence of a GSH dysregulation of genetic origin on plasma thiols (total cysteine, homocysteine, and cysteine-glycine) and other free amino acid levels as well as fibroblast cultures GSH levels. Plasma thiols levels were also compared between patients and controls. As compared with patients with a low-risk GCLC GAG TNR genotype, patients with a high-risk genotype, having an impaired GSH synthesis, displayed a decrease of fibroblast GSH and plasma total cysteine levels, and an increase of the oxidized form of cysteine (cystine) content. Increased levels of plasma free serine, glutamine, citrulline, and arginine were also observed in the high-risk genotype. Taken together, the high-risk genotypes were associated with a subgroup of schizophrenia characterized by altered plasma thiols and free amino acid levels that reflect a dysregulation of redox control and an increased susceptibility to oxidative stress. This altered pattern potentially contributes to the development of a biomarker profile useful for early diagnosis and monitoring the effectiveness of novel drugs targeting redox dysregulation in schizophrenia. Antioxid. Redox Signal. 15, 2003-2010.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles.

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.

Role of nitric oxide in genotoxicity: implication for carcinogenesis.

Reactive oxygen species can initiate carcinogenesis by virtue of their capacity to react with DNA and cause mutations. Recently, it has been suggested that nitric oxide (NO) and its derivatives produced in inflamed tissues could contribute to the carcinogenesis process. Genotoxicity of NO follows its reaction with oxygen and superoxide. It can be due either to direct DNA damage or indirect DNA damage. Direct damage includes DNA base deamination, peroxynitrite-induced adducts formation and single strand breaks in the DNA. Indirect damage is due to the interaction of NO reactive species with other molecules such as amines, thiols and lipids. The efficiency of one pathway or another might depend on the cellular antioxidant status or the presence of free metals.

Seeing smells: development of an optoelectronic nose

The development of an array of chemically-responsive dyes on a porous membrane and in its use as a general sensor for odors and volatile organic compounds (VOCs) is reviewed. These colorimetric sensor arrays (CSA) act as an "optoelectronic nose" by using an array of multiple dyes whose color changes are based on the full range of intermolecular interactions. The CSA is digitally imaged before and after exposure and the resulting difference map provides a digital fingerprint for any VOC or mixture of odorants. The result is an enormous increase in discriminatory power among odorants compared to prior electronic nose technologies. For the detection of biologically important analytes, including amines, carboxylic acids, and thiols, high sensitivities (ppbv) have been demonstrated. The array is essentially non-responsive to changes in humidity due to the hydrophobicity of the dyes and membrane.

Synthesis and biological activity of sulfur compounds showing structural analogy with combretastatin A-4

We extended our previous exploration of sulfur bridges as bioisosteric replacements for atoms forming the bridge between the aromatic rings of combretastatin A-4. Employing coupling reactions between 5-iodo-1,2,3-trimethoxybenzene and substituted thiols, followed by oxidation to sulfones with m-CPBA, different locations for attaching the sulfur atom to ring A through the synthesis of nine compounds were examined. Antitubulin activity was performed with electrophoretically homogenous bovine brain tubulin, and activity occurred with the 1,2,3-trimethoxy-4-[(4-methoxyphenyl)thio]benzene (12), while the other compounds were inactive. The compounds were also tested for leishmanicidal activity using promastigote forms of Leishmania braziliensis (MHOM/BR175/M2904), and the greatest activity was observed with 1,2,3-trimethoxy-4-(phenylthio)benzene (10) and 1,2,3-trimethoxy-4-[(4-methoxyphenyl) sulfinyl]benzene (15).

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.

Développement d'une méthode de marquage protéique par fluorescence

Le marquage protéique par fluorescence est une méthode de choix permettant d’étudier l’évolution des protéines depuis leur synthèse cellulaire jusqu’à leur dégradation, en plus de rendre possible leur localisation ainsi que la visualisation des interactions entre protéines. De cet intérêt certain ont découlé différentes techniques de marquage, dont celle présentement développée dans le groupe Keillor. Le principe de celle-ci repose sur la réaction entre deux maléimides portés par un fluorogène et une séquence peptidique cible, laquelle contient deux résidus cystéines séparés par une distance appropriée. Suite à cette double addition de thiols du peptide sur les maléimides du fluorogène, la fluorescence latente de ce dernier est régénérée, menant au marquage covalent de la protéine d’intérêt. Afin d’optimiser la spécificité et la sensibilité de cette méthode de marquage, la synthèse de nouveaux fluorogènes et l’étude de l’efficacité de quench de la fluorescence par les maléimides est présentement en cours dans les laboratoires du groupe Keillor.

Caractérisation et étude de la régulation d’une isoforme cytosolique de peroxyrédoxine chez les solanacées

Les peroxyrédoxines (PRXs) forment une famille de peroxydases communes à tous les organismes vivants et ubiquitaires dans la cellule. Leur particularité provient d’un ou deux résidus cystéines accomplissant un cycle d’oxydo-réduction à l’aide d’un donneur d’électron. Ces protéines thiols sensibles au potentiel redox sont impliquées dans le mécanisme de détoxification du H2O2, une molécule oxydante induite lors de situations de stress. Les PRXs pourraient être induites par le stress et régulées par phosphorylation. En effet, des expérimentations in vitro ont démontré que la nucléoside diphosphate kinase 1 (NDPK1) a la capacité de phosphoryler une PRX cytosolique de pomme de terre. Ce mémoire décrit les travaux expérimentaux effectués pour caractériser la fonction de la PRX. Pour cela, le clonage d’une isoforme a été effectué, suivi d’une caractérisation biochimique et d’une étude d’expression de la protéine. Les données de séquençage révèlent qu’il s’agit d’une PRX de type II phylogénétiquement liée aux PRXs cytosoliques. L’ADNc codant pour cette peroxyrédoxine (PRX1) a été cloné chez Solanum chacoense. Une protéine recombinante portant une étiquette (6xHis) en N-terminale a été produite. Des essais enzymatiques ont confirmé la fonction antioxydante de la protéine recombinante et un anticorps polyclonal a été généré chez le lapin puis utilisé en conjonction avec un anticorps anti-NDPK1 pour déterminer les patrons d’expression généraux de ces protéines chez Solanum lycopersicum et Solanum tuberosum lors de situations de stress. Les données démontrent que les deux protéines sont généralement co-exprimées mais pas co-régulées et que la PRX1 est induite en certaines situations de stress.

La bioaccumulation d’une nanoparticule d’argent (nAg) par l’algue verte Chlamydomonas reinhardtii : distinguer la contribution de la particule de celle de l’ion Ag+

L’explosion de la nanotechnologie a permis l’intégration d’une multitude de nanoparticules dans des produits de consommation. Les nanoparticules d’argent (nAg) sont les plus utilisées à ces fins, selon les derniers recensements disponibles. La plupart des études toxicologiques, à ce jour, ont fait état de l’implication très évidente de l’ion Ag+ dans la toxicité aigüe des nAg; cependant, quelques études ont mis en évidence des effets toxicologiques dus aux nAg. Il y a un certain consensus à propos d’un risque de contamination des eaux douces via leur rejet par les effluents des réseaux d’aqueducs. Puisque les concentrations en Ag+ sont généralement très faibles dans les eaux douces (de l’ordre du pg L-1), de par la formation de complexes non-labiles avec des thiols (organiques et inorganiques) et des sulfures, la toxicité inhérente aux nAg pourrait ne pas être négligeable- comparativement aux tests en laboratoires. Cette étude s’intéressait donc aux mécanismes de bioaccumulation d’argent par l’algue verte C. reinhardtii suite à l’exposition à des nAg de 5 nm (enrobage d’acide polyacrylique). La bioaccumulation d’argent pour l’exposition à Ag+ servait de point de comparaison; également, les abondances de l’ARNm de l’isocitrate lyase 1 (ICL1) et de l’ARNm de Copper Transporter 2 (CTR2) étaient mesurées comme témoins biologiques de la bioaccumulation de Ag+. Les expériences ont été menées en présence d’un tampon organique (NaHEPES, 2 x 10-2 M; Ca2+, 5x 10-5 M) à pH de 7,00. Pour des expositions à temps fixe de 2 heures, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+; cependant, il n’y avait pas de différence d’abondance des ARNm de ICL1 et de CTR2 entre nAg et Ag+. D’un autre côté, pour une exposition à temps variables, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+ et une augmentation de l’abondance de l’ARNm de ICL1 était notée pour nAg. Cependant, il n’y avait aucune différence significative au niveau de l’abondance de l’ARNm de CTR2 entre nAg et une solution équivalente en Ag+. L’ajout d’un fort ligand organique (L-Cystéine; log K= 11,5) à une solution de nAg en diminuait radicalement la bioaccumulation d’argent par rapport à nAg-sans ajout de ligand. Par contre, l’abondance des ARNm de ICL1 et de CTR2 étaient stimulées significativement par rapport à une solution contrôle non-exposée à nAg, ni à Ag+. Les résultats suggéraient fortement que les nAg généraient des ions Ag+ au contact de C. reinhardtii.

Inhibition réversible et photomarquage de la transglutaminase tissulaire

La transglutaminase tissulaire est une enzyme dépendante du calcium qui catalyse la formation de liens isopeptidiques, entre les chaînes latérales de résidus glutamine et lysine, permettant, par le fait même, la réticulation des protéines dans les systèmes biologiques. Elle joue un rôle, entre autres, dans l’endocytose, la régulation du développement des cellules, et même dans l’apoptose. Néanmoins, une dérégulation de l’activité biologique de cette enzyme peut entrainer différentes pathologies, comme la formation de cataractes, de plaques amyloïdes dans la maladie d’Alzheimer, ou encore peut mener au développement de la maladie céliaque. C’est pourquoi une meilleure connaissance du mécanisme d’action de cette enzyme et la possibilité de réguler son action à l’aide de substrats ou d’inhibiteurs sont nécessaires. Dans cette optique, une méthode d’expression et de purification de la transglutaminase humaine a été développée, permettant de travailler directement avec la cible pharmacologique désirée. De plus, une étude du mode d’inhibition et de liaison d’une classe d’inhibiteurs réversibles précédemment découverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d’identifier que la puissance de ces molécules est influencée par la présence du calcium et qu’une inhibition dépendante du temps est observée, en lien avec un potentiel équilibre conformationnel lent de la transglutaminase. D’un autre côté, la susceptibilité à une attaque nucléophile par des thiols de cette classe de molécule rend leur potentiel pharmacologique grandement diminué, et c’est pourquoi une nouvelle famille de molécules a été identifiée, basée sur un squelette ynone, avec une valeur d’IC50 très prometteuse de 2,6 μM, en faisant un des meilleurs inhibiteurs réversibles de la transglutaminase développés à ce jour. Finalement, une stratégie de photomarquage jumelée à une analyse de spectrométrie de masse en tandem a été développée pour la découverte du site de liaison du substrat dérivé de la lysine, dans le but de mieux comprendre le mécanisme complexe de cette enzyme.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.