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It has been shown that the alternative oxidase in mitochondria of fungi and plants has important functions in the response against stress conditions, although their role in some organisms is still unknown. This is the case of

Reduced glutathione (GSH) is the most abundant non-protein endogenous thiol. It is a ubiquitous molecule produced in most organs, but its synthesis is predominantly in the liver, the tissue in charge of storing and distributing it. GSH is involved in the detoxification of free radicals, peroxides and xenobiotics (drugs, pollutants, carcinogens, etc.), protects biological membranes from lipid peroxidation, and is an important regulator of cell homeostasis, since it participates in signaling redox, regulation of the synthesis and degradation of proteins (S-glutathionylation), signal transduction, various apoptotic processes, gene expression, cell proliferation, DNA and RNA synthesis, etc. GSH transport is a vital step in cellular homeostasis supported by the liver through providing extrahepatic organs (such as the kidney, lung, intestine, and brain, among others) with the said antioxidant. The wide range of functions within the cell in which glutathione is involved shows that glutathione’s role in cellular homeostasis goes beyond being a simple antioxidant agent; therefore, the importance of this tripeptide needs to be reassessed from a broader metabolic perspective.

It has been shown that the alternative oxidase in mitochondria of fungi and plants has important functions in the response against stress conditions, although their role in some organisms is still unknown. This is the case of Ustilago maydis. There is no evidence of the participation of the U. maydis Aox1 in stressful conditions such as desiccation, high or low temperature, and low pH, among others. Therefore, in this work, we studied the role of the U. maydis Aox1 in cells exposed to oxidative stress induced by methyl viologen (paraquat). To gain insights into the role of this enzyme, we took advantage of four strains: the FB2 wild-type, a strain without the alternative oxidase (FB2aox1Δ), other with the Aox1 fused to the Gfp under the control of the original promoter (FB2aox1-Gfp), and one expressing constitutively de Aox1-Gfp (FB2Potef:aox1-Gfp). Cells were incubated for various times in the presence of 1 mM paraquat and growth, replicative capacities, mitochondrial respiratory activity, Aox1 capacity, and the activities of several antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase) were assayed. The results show that (1) the response of U. maydis against oxidative stress was the same in the presence or absence of the Aox1; (2) the activities of the antioxidant enzymes remained constant despite the oxidative stress; and (3) there was a decrease in the GSH/GSSG ratio in U. maydis cells incubated with paraquat.

Type 2 alternative NADH dehydrogenases (NDH‐2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH‐2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH‐2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

Cardiovascular diseases (CVD) (such as occlusion of the coronary arteries, hypertensive heart diseases and strokes) are diseases that generate thousands of patients with a high mortality rate worldwide. Many of these cardiovascular pathologies, during their development, generate a state of oxidative stress that leads to a deterioration in the patient’s conditions associated with the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Within these reactive species we find superoxide anion (O2•–), hydroxyl radical (•OH), nitric oxide (NO•), as well as other species of non-free radicals such as hydrogen peroxide (H2O2), hypochlorous acid (HClO) and peroxynitrite (ONOO–). A molecule that actively participates in counteracting the oxidizing effect of reactive species is reduced glutathione (GSH), a tripeptide that is present in all tissues and that its synthesis and/or regeneration is very important to be able to respond to the increase in oxidizing agents. In this review, we will address the role of glutathione, its synthesis in both the heart and the liver, and its importance in preventing or reducing deleterious ROS effects in cardiovascular diseases.

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

Acute ethanol intoxication increased triacylglycerides (TAG) and thiobarbituric acid reactive substances (TBARS) in liver and promoted the liberation of epinephrine. Four non-steroidal anti-inflammatory drugs (NSAIDs) - aspirin, naproxen, nimesulide and piroxicam - prevented this increase in TAG and TBARS. Because fatty acids provided by adipose tissue contribute substantially to elevated hepatic TAG in ethanol-intoxicated rats, it was thought that the NSAIDs might reduce epinephrine-stimulated lipolysis in these rats. Isolated rat adipocytes were activated with epinephrine in the presence or absence of the NSAIDs. The NSAIDs inhibited epinephrine-stimulated lipolysis. These drugs did not modify the binding of dihydroalprenolol (β-adrenergic agonist) to their receptors in isolated guinea-pig liver membranes. The NSAIDs, at concentrations 3000-fold lower than that of cAMP, inhibited stimulated lipolysis by this messenger. In conclusion, aspirin, naproxen, nimesulide and piroxicam reduce the release of fatty acids from adipose tissue to the liver by inhibiting the epinephrine-stimulated lipolysis, and this, in part, explains the protective action of these NSAIDs against hepatic signs of acute ethanol intoxication.

Hepatic surgery requires, under diverse circumstances, periods of ischemia and reperfusion (I-R) such as those present in liver resection, hepatic injury, and liver transplantation. The objective of the present work was to conduct an experimental study to evaluate the effect of hepatic preconditioning (HPC) on modulation of the I-R injury.Male Wistar rats were distributed into the following three study groups: group 1, simulated or sham; group 2, submitted to a 30-min period of total warm ischemia and a reperfusion phase, and group 3, in which we carried out 10-min preconditioning of warm ischemia and 10 min of reperfusion prior to the total ischemia period for a total of 60 min and the reperfusion phase. We obtained liver biopsies for thiobarbituric acid reactive substances (TBARS) (MDA, thiobarbituric acid adducts) and for blood sample determinations in serum of liver-cell enzymes such as alanine aminotransferase and aspartate aminotransferase during a 24-h time course.We observed a decrease in the variables studied in group 3 (HPC) as well as of serum liver enzymes and TBARS levels such as indirect oxidative stress indicators upon comparison with group 2 animals submitted to total hepatic I-R.HPC is an efficient surgical strategy for decreasing the elevation of hepatic enzymes and indirect lipoperoxidation indicators in an I-R model. Controlled clinical studies should be performed to determine its functional properties and clinical applicability.

Among many actions assigned to taurine (Tau), the most abundant amino acid in numerous mammalian tissues, it prevents high-fat diet-induced obesity with increasing resting energy expenditure. To sustain this Tau action, the goal of the present study was to explore the acute effects of Tau on baseline and on adrenaline, insulin and their second messengers to modulate lipolysis in white adipose tissue (WAT) cells from rat epididymis. The Tau effects in this topic were compared with those recorded with Gly, Cys and Met. Tau on its own did not modify baseline lipolysis. Tau raised isoproterenol- and dibutyryl-cAMP (Bt2cAMP)-activated glycerol release. Gly diminished Bt2cAMP-activated glycerol release, and Cys and Met had no effect. Cyclic AMP-dependent activation of protein kinase A (PKA) in cell-free extracts decreased slightly by Gly and was unaltered by Cys, Met, and Tau. PKA catalytic activity in cell-free extracts was stimulated by Tau and unchanged by Cys, Gly and Met. Gly and Tau effects on PKA disappeared when these amino acids were withdrawn by gel filtration. Insulin-mediated NADPH-oxidase (NOX) raises H2O2 pool, which promotes PKA subunit oxidation, and precludes its cAMP activation; thus, lipolysis is diminished. Tau, but not Cys, Gly and Met, inhibited, by as much as 70%, insulin-mediated H2O2 pool increase. These data suggested that Tau raised lipolysis in adipocytes by two mechanisms: stimulating cAMP-dependent PKA catalytic activity and favoring PKA activation by cAMP as a consequence of lowering the H2O2 pool.

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

1. Non-steroidal anti-inflammatory drugs (NSAIDs) [acetylsalicylic acid (ASS), naproxen, nimesulide and piroxicam] decreased adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes. We aimed to determine the mechanism of this NSAIDs action. 2. Non-steroidal anti-inflammatory drugs decreased cAMP-dependent protein kinase A (PKA) activity in rat adipocyte lysates and in a commercial bovine heart PKA holoenzyme. If added before cAMP, NSAIDs impaired PKA activation by the cyclic nucleotide; however, if PKA was first activated by cAMP, NSAIDs were ineffective. NSAIDs were also ineffective against PKA catalytic subunits. 3. Consequently, NSAIDs lowered hormone-sensitive lipase translocation from cytosol to lipid storage droplets in adipocytes lysates, the critical event to promote lipolysis. 4. These results indicate that inhibition of PKA activation explains NSAIDs-induced decrease in adrenaline-stimulated lipolysis. We suggest that reproduction of such inhibition in nociceptive cells might enhance the understanding of the mechanism underlying the analgesic effects of NSAIDs.