How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The presence of the NOS3 gene polymorphism for intron 4 mitigates the beneficial effects of exercise training on ambulatory blood pressure monitoring in adults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1 mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5 mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (similar to 2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Thin functional conducting polymer films

In the present study, thin functional conducting polyaniline (PANI) films, either doped or undoped, patterned or unpatterned, were prepared by different approaches. The properties of the obtained PANI films were investigated in detail by a combination of electrochemistry with several other techniques, such as SPR, QCM, SPFS, diffraction, etc. The sensing applications (especially biosensing applications) of the prepared PANI films were explored. Firstly, the pure PANI films were prepared by the electropolymerisation method and their doping/dedoping properties in acidic conditions were investigated in detail by a combination of electrochemistry with SPR and QCM. Dielectric constants of PANI at different oxidation states were obtained quantitatively. The results obtained here laid a good foundation for the following investigations of PANI films in neutral pH conditions. Next, PANI multilayer films doped by a variety of materials were prepared by the layer-by-layer method in order to explore their biosensing applications, because of the loss of redox activity of pure PANI in neutral pH conditions. The dopants used include not only the traditionally used linear polyelectrolytes, but also, for the first tim, some other novel materials, like modified gold nanoparticles or modified carbon nanotubes. Our results showed that all the used dopants could form stable multilayer films with PANI. All the obtained PANI multilayer films showed good redox activity in a neutral pH environment, which makes them feasible for bioassays. We found that all the prepared PANI multilayer films can electrocatalyze the oxidation of NADH in neutral conditions at a low potential, although their catalytic efficiencies are different. Among them, PANI/carbon nanotube system showed the highest catalytic efficiency toward the oxidation of NADH, which makes it a good candidate as a NADH sensor. Besides, because some of the prepared PANI multilayer systems were end-terminated with –COOH groups (like PANI/Au nanoparticles system), which can be utilized to easily link biomolecules for biosensing applications. Here, we demonstrated, for the first time, to use the prepared PANI multilayer films for the DNA hybridisation detection. The detection event was monitored either by direct electrochemical method, or by enzyme-amplified electrochemical method, or by surface plasmon enhanced fluorescence spectroscopic method. All the methods can effectively differentiate non-complementary DNA from the complementary ones, even at the single-base mismatch level. It should also be noted that, our success in fabricating PANI multilayer films with modified Au nanoparticles or carbon nanotubes also offered another novel method for incorporating such novel materials into (conducting) polymers. Because of the unique electrochemical and optical properties of each component of the obtained PANI multilayer films, they should also find potential applications in many other fields such as microelectronics, or for electrochromic and photovoltaic devices. Finally, patterned PANI films were fabricated by the combination of several patterning techniques, such as the combination of electrocopolymerization with micromolding in capillaries (EP-MIMIC), the combination of microcontact printing with the layer-by-layer technique (µCP-LBL), and the polystyrene (PS) template induced electropolymerisation method. Using the obtained stripe-shaped PANI/PSS film, a redox-switchable polymer grating based on the surface-plasmon-enhanced mode was constructed and its application in the field of biosensing was explored. It was found that the diffraction efficiency (DE) of the grating was very sensitive to the applied potential (i.e. redox state of the film) as well as the pH environment of the dielectric medium. Moreover, the DE could also be effectively tuned by an electrocatalytic event, such as the electrocatalytic oxidation of NADH by the grating film. By using PS colloidal crystal assemblies as templates, well-ordered 3D interconnected macroporous PANI arrays (PANI inverse opals) were fabricated via electropolymerisation method. The quality of the obtained inverse opals was much higher than those reported by chemical synthesis method. By electrochemical method, the structures of the prepared inverse opals can be easily controlled. To explore the possible biosensing applications of PANI inverse opals, efforts were also done toward the fabrication of PANI composite inverse opals. By selecting proper dopants, high quality inverse opals of PANI composites were fabricated for the first time. And the obtained opaline films remained redox-active in neutral pH conditions, pointing to their possible applications for electrobioassays.

Sulforaphane as a multifunctional neuroprotective molecule to prevent and slow down the progression of Alzheimer’s disease

Oxidative stress has been implicated in the pathogenesis of a number of diseases including neurodegenerative disorders, cancer, ischemia, etc. Alzheimer’s disease (AD) is histopathologically characterized by the presence of extracellular senile plaque (SP), predominantly consisting of fibrillar amyloid-peptide (Aβ), intracellular neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau protein, and cell loss in the selected regions of the brain. However, the pathogenesis of AD remains largely unknown, but a number of hypothesis were proposed for AD mechanisms, which include: the amyloid cascade, excitotoxicity, oxidative stress and inflammation hypothesis, and all of them are based, to some extent on the role of A. Accumulated evidence indicates that the increased levels of ROS may act as important mediators of synaptic loss and eventually promote formation of neurofibrillary tangles and senile plaques. Therefore a vicious circle between ROS and Aaccumulation may accelerate progression of AD. For these reasons, growing attention has focused on oxidative mechanism of Atoxicity as well as the search for novel neuroprotective agents. A strategy to prevent the oxidative stress in neurons may be the use of chemopreventive agents as inducers of antioxidant and phase 2 enzymes. Sulforaphane (SF), derived from corresponding glucoraphanin, glucosinolate found in abundance in cruciferous vegetables, has recently gained attention as a potential neuroprotective compound inducer of antioxidant phase 2 enzymes. Consistent with this evidence, the study is aimed at identifying the SF ability to prevent and counteract the oxidative damage inducted by oligomers of Aβ (1-42) in terms of impairment in the intracellular redox state and cellular death in differentiated human neuroblastoma and microglia primary cultures. In addition we will evaluated the mechanism underlying the SF neuroprotection activity.

Ein neues Lumineszenzverfahren zur quantitativen und räumlichen Darstellung von Produkten des Glukosestoffwechsels in Tumoren

Eine neue auf einer Pyruvat abhängigen Biolumineszenzreaktion basierende Methode zur quantitativen Bestimmung und räumlichen Darstellung von Pyruvat in Gefrierschnitten von Gewebeproben wurde entwickelt. Dabei wurden biochemische Reaktionen so verknüpft, dass sichtbares Licht proportional zum eingesetzten Pyruvatgehalt entstand. Eine hoch signifikante positive Korrelation beider Parameter ermöglichte eine Kalibrierung mit definierten Pyruvatgehalten und damit die Quantifizierung in unbekannten Proben. Die Nachweisgrenze lag bei 0,04 pmol Pyruvat mit einer Auflösung von 0,02 µmol/g. Das Biolumineszenzverfahren wurde mit Hilfe anderer Methoden validiert, wobei eine Wiederfindung mit einer konzentrationsabhängigen Abweichung von ≤ 15 % erzielt wurde. Ein wesentlicher Vorteil der neuen Methode gegenüber bisherigen Verfahren zum Pyruvatnachweis liegt in der Messwerterfassung definierter histologischer Gewebsareale. Dies wird durch computergesteuerte Überlagerung von Metabolitverteilungen mit Schnittbildern aus Strukturfärbungen und interaktiver, „optischer Mikrodissektion“ der Gewebeschnitte möglich. Ein weiterer Nutzen der Methode ist deren optionale Kombination mit der Biolumineszenztechnik für andere Stoffwechselprodukte. So ermöglicht eine exakte Superposition zweier Metabolitbilder von unmittelbar aufeinander folgenden Gewebeschnitten eine korrelative Kolokalisationsanalyse beider Metabolite. Das Ergebnis lässt sich zum einen in Form von „Pixel-zu-Pixel“-Korrelationen dokumentieren, zum anderen kann für jeden Bildpunkt ein Laktat/Pyruvat-Verhältnis als Maß für den Redoxzustand des Gewebes berechnet und dargestellt werden. Hieraus ergeben sich z.B. räumliche L/P-Verteilungen (L/P-Karten). Ein solches „Redoximaging“ durch Kartierung des L/P-Quotienten ist bislang mit keinem anderen Verfahren möglich. Während die Entwicklung des Pyruvatnachweises eine Kernaufgabe der vorliegenden Arbeit darstellte, bestand ein weiterer wesentlicher Teil in der praktischen Anwendung der neuen Methode im Bereich der experimentellen Tumorforschung. So ergaben Messungen an acht verschiedenen Linien von humanen HNSCC-Xenotransplantaten (n = 70 Tumoren) einen mittleren Pyruvatgehalt von 1,24 ± 0,20 µmol/g. In sechs Humanbiopsien derselben Tumorentität wurde ein durchschnittlicher Pyruvatgehalt von 0,41 ± 0,09 µmol/g gemessen. Bei den Xenotransplantaten konnte eine signifikante positive Korrelation zwischen der Summe aus Laktat und Pyruvat bzw. dem L/P Verhältnis und der Strahlensensibilität gefunden werden, wobei das L/P-Verhältnis ebenso wie die Summe aus Laktat und Pyruvat maßgeblich von Laktat bestimmt wurden. Der Zusammenhang der Metabolite mit der Strahlensensibilität lässt sich durch deren antioxidative Eigenschaften erklären. Da der Redoxzustand der Zelle kritisch bezüglich der Effizienz von ROS induzierenden Therapieansätzen, wie z.B. Bestrahlung oder bestimmter Chemotherapeutika sein kann, könnte die Bestimmung des L/P Verhältnisses als prognostischer Faktor prädiktive Aussagen über die Sensibilität gegenüber solchen Behandlungen erlauben.

Decreased infarct size after focal cerebral ischemia in mice chronically infected with Toxoplasma gondii

To determine whether Toxoplasma gondii infection could modify biological phenomena associated with brain ischemia, we investigated the effect of permanent middle cerebral artery occlusion (MCAO) on neuronal survival, inflammation and redox state in chronically infected mice. Infected animals showed a 40% to 50% decrease of infarct size compared with non-infected littermates 1, 4 and 14 days after MCAO. The resistance of infected mice may be associated with increased basal levels of anti-inflammatory cytokines and/or a marked reduction of the MCAO-related brain induction of two pro-inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma (IFNgamma). In addition, potential anti-inflammatory/neuroprotective factors such as nerve growth factor, suppressor of cytokine signaling-3, superoxide dismutase activity, uncoupling protein-2 and glutathione (GSH) were upregulated in the brain of infected mice. Consistent with a role of GSH in central cytokine regulation, GSH depletion by diethyl maleate inhibited Toxoplasma gondii lesion resistance by increasing the proinflammatory cytokine IFNgamma brain levels. Overall, these findings indicate that chronic toxoplasmosis decisively influences both the inflammatory molecular events and outcome of cerebral ischemia.

Electron transport through catechol-functionalized molecular rods

The charge transport properties of a catechol-type dithiol-terminated oligo-phenylene-ethynylene was investigated by cyclic voltammetry (CV) and by the scanning tunnelling microscopy break junction technique (STM-BJ). Single molecule charge transport experiments demonstrated the existence of high and low conductance regions. The junction conductance is rather weakly dependent on the redox state of the bridging molecule. However, a distinct dependence of junction formation probability and of relative stretching distances of the catechol- and quinone-type molecular junctions is observed. Substitution of the central catechol ring with alkoxy-moieties and the combination with a topological analysis of possible π-electron pathways through the respective molecular skeletons lead to a working hypothesis, which could rationalize the experimentally observed conductance characteristics of the redox-active nanojunctions.

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

Electrochemical control of a non-covalent binding between ferrocene and beta-cyclodextrin

The forces required for the detachment of ferrocene (Fc) from β-cyclodextrin (βCD) in a single host (βCD)–guest (Fc) complex were investigated using force spectroscopy under electrochemical conditions. The redox state of the guest Fc moiety as well as the structure of the supporting matrix was found to decisively affect the nanomechanical properties of the complex.

Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Magnetic susceptibility and electric conductivity of marine surficial sediments by benthic electromagnetic profiling

Distribution, accumulation and diagenesis of surficial sediments in coastal and continental shelf systems follow complex chains of localized processes and form deposits of great spatial variability. Given the environmental and economic relevance of ocean margins, there is growing need for innovative geophysical exploration methods to characterize seafloor sediments by more than acoustic properties. A newly conceptualized benthic profiling and data processing approach based on controlled source electromagnetic (CSEM) imaging permits to coevally quantify the magnetic susceptibility and the electric conductivity of shallow marine deposits. The two physical properties differ fundamentally insofar as magnetic susceptibility mostly assesses solid particle characteristics such as terrigenous or iron mineral content, redox state and contamination level, while electric conductivity primarily relates to the fluid-filled pore space and detects salinity, porosity and grain-size variations. We develop and validate a layered half-space inversion algorithm for submarine multifrequency CSEM with concentric sensor configuration. Guided by results of modeling, we modified a commercial land CSEM sensor for submarine application, which was mounted into a nonconductive and nonmagnetic bottom-towed sled. This benthic EM profiler Neridis II achieves 25 soundings/second at 3-4 knots over continuous profiles of up to hundred kilometers. Magnetic susceptibility is determined from the 75 Hz in-phase response (90% signal originates from the top 50 cm), while electric conductivity is derived from the 5 kHz out-of-phase (quadrature) component (90% signal from the top 92 cm). Exemplary survey data from the north-west Iberian margin underline the excellent sensitivity, functionality and robustness of the system in littoral (~0-50 m) and neritic (~50-300 m) environments. Susceptibility vs. porosity cross-plots successfully identify known lithofacies units and their transitions. All presently available data indicate an eminent potential of CSEM profiling for assessing the complex distribution of shallow marine surficial sediments and for revealing climatic, hydrodynamic, diagenetic and anthropogenic factors governing their formation.

Geochemistry of ODP Site 145-882

Measurements of benthic foraminiferal cadmium:calcium (Cd/Ca) have indicated that the glacial-interglacial change in deep North Pacific phosphate (PO4) concentration was minimal, which has been taken by some workers as a sign that the biological pump did not store more carbon in the deep glacial ocean. Here we present sedimentary redox-sensitive trace metal records from Ocean Drilling Program (ODP) Site 882 (NW subarctic Pacific, water depth 3244 m) to make inferences about changes in deep North Pacific oxygenation - and thus respired carbon storage - over the past 150,000 yr. These observations are complemented with biogenic barium and opal measurements as indicators for past organic carbon export to separate the influences of deep-water oxygen concentration and sedimentary organic carbon respiration on the redox state of the sediment. Our results suggest that the deep subarctic Pacific water mass was depleted in oxygen during glacial maxima, though it was not anoxic. We reconcile our results with the existing benthic foraminiferal Cd/Ca by invoking a decrease in the fraction of the deep ocean nutrient inventory that was preformed, rather than remineralized. This change would have corresponded to an increase in the deep Pacific storage of respired carbon, which would have lowered atmospheric carbon dioxide (CO2) by sequestering CO2 away from the atmosphere and by increasing ocean alkalinity through a transient dissolution event in the deep sea. The magnitude of change in preformed nutrients suggested by the North Pacific data would have accounted for a majority of the observed decrease in glacial atmospheric pCO2.