Calorie restriction increases cerebral mitochondrial respiratory capacity in a NO center dot-mediated mechanism: Impact on neuronal survival

Calorie restriction (CR) enhances animal life span and prevents age-related diseases, including neurological decline. Recent evidence suggests that a mechanism involved in CR-induced life-span extension is NO-stimulated mitochondrial biogenesis. We examine here the effects of CR on brain mitochondrial content. CR increased eNOS and nNOS and the content of mitochondria] proteins (cytochrome c oxidase, citrate synthase, and mitofusin) in the brain. Furthermore, we established an in vitro system to study the neurological effects of CR using serum extracted from animals on this diet. In cultured neurons, CR serum enhanced nNOS expression and increased levels of nitrite (a NO product). CR serum also enhanced the levels of cytochrome c oxidase and increased citrate synthase activity and respiratory rates in neurons. CR serum effects were inhibited by L-NAME and mimicked by the NO donor SNAP. Furthermore, both CR sera and SNAP were capable of improving neuronal survival. Overall, our results indicate that CR increases mitochondrial biogenesis in a NO-mediated manner, resulting in enhanced reserve respiratory capacity and improved survival in neurons. (C) 2012 Elsevier Inc. All rights reserved.

Purificação e caracterização da VDAC de mitocôndrias corticais aviares: identificação de modificações pós-traducionais nas porinas neuronais murinas e aviares

A VDAC é uma porina presente na MME cuja função é crucial no metabolismo energético, sobrevivência e morte celular. A caracterização da VDAC torna-se importante para a compreensão das inter-relações da mitocôndria com os diferentes componentes citosólicos, tais como a HK. A ligação HK-VDAC favorece a utilização do ATP intramitocondrial em células neuronais, a HK cerebral pode interagir de formas diferentes com a VDAC, o que resulta em diferentes sítios de ligação (sítios A e B). Os variados papéis metabólicos das isoformas da VDAC podem ser explicados pela presença de alterações pós-traducionais. No presente trabalho purificamos a VDAC1 mitocondrial neuronal proveniente de cérebro aviar. Paralelamente, comprovamos que a presença de múltiplas formas das VDACs 1 e 2 em cérebros murino e aviar, seja devida à presença de modificações pós-traducionais, nomeadamente a fosforilação. A proteína isolada apresentou peso molecular de 30KDa. Quando submetida à eletroforese e posteriormente à coloração para a identificação de fosfoproteínas, a mesma mostrou-se desfosforilada. O conhecimento da presença, ou ausência de fosforilação das VDACs, reside na importância de estabelecer-se as bases moleculares ligadas à existência de sítios A e B nas mitocôndrias neuronais.

Tamoxifen Subcellular Localization; Observation of Cell-Specific Cytotoxicity Enhancement by Inhibition of Mitochondrial ETC Complexes I and III

Recently, a nongenomic cytotoxic component of the chemotherapeutic agent tamoxifen (TAM) has been identified that predominantly triggers mitochondrial events. The present study delineates the intracellular fate of TAM and studies its interaction with a spectrum of cell homeostasis modulators primarily relevant to mitochondria. The subcellular localization of TAM was assessed by confocal fluorescence microscopy. The effect of the modulators on TAM cytotoxicity was assessed by standard MTT assays. Our findings show that in estrogen receptor positive MCF7 breast adenocarcinoma cells and DU145 human prostate cancer cells, TAM largely accumulates in the mitochondria and endoplasmic reticulum, but not lysosomes. Our results further demonstrate that in MCF7, but not in DU145 cells, mitochondrial electron transport chain complex I and III inhibitors exacerbate TAM toxicity with an order of potency of myxothiazol = stigmatellin > rotenone > antimycin A, suggesting a cell-specific cytotoxic interplay between mitochondrial complex I and III function and TAM action.

Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

Nitroglycerin (GIN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GIN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GIN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1-50 nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP(3), probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GIN pharmacological action at pharmacologically relevant doses. (C) 2011 Elsevier Inc. All rights reserved.

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Metalloporphyrins as cytochrome P450 models for chlorhexidine metabolite prediction

The catalytic oxidation of chlorhexidine (CHX, a strong microbicidal agent) mediated by ironporphyrins has been investigated by using hydrogen peroxide, mCPBA, tBuOOH, or NaOCl as oxidant. All of these oxygen donors yielded p-chloroaniline (pCA) as the main product. The higher pCA yields amounted to 71% in the following conditions: catalyst/oxidant/substrate molar ratio of 1:150:50, aqueous medium, FeTMPyP as catalyst. The medium pH also had a strong effect on the pCA yields; in physiological pH, formation of this product was specially favored in the presence of the catalysts, with yields 58% higher than those achieved in control reactions. This provided strong evidence that CHX is metabolized to pCA upon ingestion. (c) 2012 Elsevier B.V. All rights reserved.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO2)(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO2 formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca2+-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,45,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes. (C) 2012 Elsevier Inc. All rights reserved.

Photo-induced electron transfer in supramolecular materials of titania nanostructures and cytochrome c

In the present paper, we report on the molecular interaction and photochemistry of TiO2 nanoparticles (NPs) and cytochrome c systems for understanding the effects of supramolecular organization and electron transfer by using two TiO2 structures: P25 TiO2 NPs and titanate nanotubes. The adsorption and reduction of cytochrome c heme iron promoted by photo-excited TiO2, arranged as P25 TiO2 NPs and as nanotubes, were characterized using electronic absorption spectroscopy, thermogravimetric analysis, and atomic force microscopy. In an aqueous buffered suspension (pH 8.0), the mass of cytochrome c adsorbed on the P25 TiO2 NP surface was 2.3 fold lower (0.75 mu g m(-2)) than that adsorbed on the titanate nanotubes (1.75 mu g m(-2)). Probably due to the high coverage of titanate nanotubes by adsorbed cytochrome c, the low amount of soluble remaining protein was not as efficiently photo-reduced by this nanostructure as it was by the P25 TiO2 NPs. Cytochrome c, which desorbed from both titanium materials, did not exhibit changes in its redox properties. In the presence of the TiO2 NPs, the photo-induced electron transfer from water to soluble cytochrome c heme iron was corroborated by the following findings: (i) identification by EPR of the hydroxyl radical production during the irradiation of an aqueous suspension of TiO2 NPs, (ii) impairment of a cytochrome c reduction by photo-excited TiO2 in the presence of dioxane, which affects the dielectric constant of the water, and (iii) change in the rate of TiO2-promoted cytochrome c reduction when water was replaced with D2O. The TiO2-promoted photo-reduction of cytochrome c was reverted by peroxides. Cytochrome c incorporated in the titanate nanotubes was also reversibly reduced under irradiation, as confirmed by EPR and UV-visible spectroscopy.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

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.