TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation.

It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

L-selectin expression is regulated by CXCL8-induced reactive oxygen species produced during human neutrophil rolling.

Neutrophils destroy invading microorganisms by phagocytosis by bringing them into contact with bactericidal substances, among which ROS are the most important. However, ROS also function as important physiological regulators of cellular signaling pathways. Here, we addressed the involvement of oxygen derivatives in the regulation of human neutrophil rolling, an essential component of the inflammatory response. Flow experiments using dihydroethidium-preloaded human neutrophils showed that these cells initiate an early production of intracellular ROS during the rolling phase of the adhesion cascade, a phenomenon that required cell rolling, and the interaction of the chemokine receptor CXCR2 with their ligand CXCL8. Flow cytometry experiments demonstrated that L-selectin shedding in neutrophils is triggered by ROS through an autocrine-paracrine mechanism. Preincubation of neutrophils with the NADPH oxidase complex inhibitor diphenyleniodonium chloride significantly increased the number of rolling neutrophils on endothelial cells. Interestingly, the same effect was observed when CXCL8 signaling was interfered using either a blocking monoclonal antibody or an inhibitor of its receptor. These findings indicate that, in response to CXCL8, neutrophils initiate ROS production during the rolling phase of the inflammatory response. This very early ROS production might participate in the modulation of the inflammatory response by inducing L-selectin shedding in neutrophils.

eNOS S-nitrosylates ß-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of ß-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated ß-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of ß-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca2+ concentration ([Ca2+ ]c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca2+ ]c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.

The APPswe/PS1A246E mutations in an astrocytic cell line leads to increased vulnerability to oxygen and glucose deprivation, Ca2+ dysregulation, and mitochondrial abnormalities.

Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APPSWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APPSWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca2+ concentration ([Ca2+ ]c ) were potentiated in the APPSWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APPSWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

A novel strategy for global analysis of the dynamic thiol redox proteome.

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

The role of ferroptosis as a regulator of oxidative stress in the pathogenesis of ischemic stroke.

Ferroptosis is a unique form of cell death that was first described in 2012 and plays a significant role in various diseases, including neurodegenerative conditions. It depends on a dysregulation of cellular iron metabolism, which increases free, redox-active, iron that can trigger Fenton reactions, generating hydroxyl radicals that damage cells through oxidative stress and lipid peroxidation. Lipid peroxides, resulting mainly from unsaturated fatty acids, damage cells by disrupting membrane integrity and propagating cell death signals. Moreover, lipid peroxide degradation products can further affect cellular components such as DNA, proteins, and amines. In ischemic stroke, where blood flow to the brain is restricted, there is increased iron absorption, oxidative stress, and compromised blood-brain barrier integrity. Imbalances in iron-transport and -storage proteins increase lipid oxidation and contribute to neuronal damage, thus pointing to the possibility of brain cells, especially neurons, dying from ferroptosis. Here, we review the evidence showing a role of ferroptosis in ischemic stroke, both in recent studies directly assessing this type of cell death, as well as in previous studies showing evidence that can now be revisited with our new knowledge on ferroptosis mechanisms. We also review the efforts made to target ferroptosis in ischemic stroke as a possible treatment to mitigate cellular damage and death.

The mitochondrial Na+/Ca2+ exchanger NCLX is implied in the activation of hypoxia-inducible factors.

Eukaryotic cells and organisms depend on oxygen for basic living functions, and they display a panoply of adaptations to situations in which oxygen availability is diminished (hypoxia). A number of these responses in animals are mediated by changes in gene expression programs directed by hypoxia-inducible factors (HIFs), whose main mechanism of stabilization and functional activation in response to decreased cytosolic oxygen concentration was elucidated two decades ago. Human acute responses to hypoxia have been known for decades, although their precise molecular mechanism for oxygen sensing is not fully understood. It is already known that a redox component, linked with reactive oxygen species (ROS) production of mitochondrial origin, is implied in these responses. We have recently described a mechanism by which the mitochondrial sodium/calcium exchanger, NCLX, participates in mitochondrial electron transport chain regulation and ROS production in response to acute hypoxia. Here we show that NCLX is also implied in the response to hypoxia mediated by the HIFs. By using a NCLX inhibitor and interference RNA we show that NCLX activity is necessary for HIF-α subunits stabilization in hypoxia and for HIF-1-dependent transcriptional activity. We also show that hypoxic mitochondrial ROS production is not required for HIF-1α stabilization under all circumstances, suggesting that the basal cytosolic redox state or other mechanism(s) could be operating in the NCLX-mediated response to hypoxia that operates through HIF-α stabilization. This finding provides a link between acute and medium-term responses to hypoxia, reinforcing a central role of mitochondrial cell signalling in the response to hypoxia.

Inhibition of ALK3-mediated signalling pathway protects against acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is one of the most prevalent causes of acute liver failure (ALF). We assessed the role of the bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 in APAP-induced hepatotoxicity. The molecular mechanisms that regulate the balance between cell death and survival and the response to oxidative stress induced by APAP was assessed in cultured human hepatocyte-derived (Huh7) cells treated with pharmacological inhibitors of ALK receptors and with modulated expression of ALK2 or ALK3 by lentiviral infection, and in a mouse model of APAP-induced hepatotoxicity. Inhibition of ALK3 signalling with the pharmacological inhibitor DMH2, or by silencing of ALK3, showed a decreased cell death both by necrosis and apoptosis after APAP treatment. Also, upon APAP challenge, ROS generation was ameliorated and, thus, ROS-mediated JNK and P38 MAPK phosphorylation was reduced in ALK3-inhibited cells compared to control cells. These results were also observed in an experimental model of APAP-induced ALF in which post-treatment with DMH2 after APAP administration significantly reduced liver tissue damage, apoptosis and oxidative stress. This study shows the protective effect of ALK3 receptor inhibition against APAP-induced hepatotoxicity. Furthermore, findings obtained from the animal model suggest that BMP signalling might be a new pharmacological target for the treatment of ALF.

Regulation of Ras Signaling by S-Nitrosylation.

Ras are a family of small GTPases that function as signal transduction mediators and are involved in cell proliferation, migration, differentiation and survival. The significance of Ras is further evidenced by the fact that Ras genes are among the most mutated oncogenes in different types of cancers. After translation, Ras proteins can be targets of post-translational modifications (PTM), which can alter the intracellular dynamics of the protein. In this review, we will focus on how S-nitrosylation of Ras affects the way these proteins interact with membranes, its cellular localization, and its activity. S-Nitrosylation occurs when a nitrosyl moiety of nitric oxide (NO) is covalently attached to a thiol group of a cysteine residue in a target protein. In Ras, the conserved Cys118 is the most surface-exposed Cys and the preferable residue for NO action, leading to the initiation of transduction events. Ras transduces the mitogen-activated protein kinases (MAPK), the phosphoinositide-3 kinase (PI3K) and the RalGEF cellular pathways. S-Nitrosylation of elements of the RalGEF cascade remains to be identified. On the contrary, it is well established that several components of the MAPK and PI3K pathways, as well as different proteins associated with these cascades, can be modified by S-nitrosylation. Overall, this review presents a better understanding of Ras S-nitrosylation, increasing the knowledge on the dynamics of these proteins in the presence of NO and the underlying implications in cellular signaling.

Importance of immunometabolic markers for the classification of patients with major depressive disorder using machine learning.

BACKGROUND: Although there is scientific evidence of the presence of immunometabolic alterations in major depression, not all patients present them. Recent studies point to the association between an inflammatory phenotype and certain clinical symptoms in patients with depression. The objective of our study was to classify major depression disorder patients using supervised learning algorithms or machine learning, based on immunometabolic and oxidative stress biomarkers and lifestyle habits. METHODS: Taking into account a series of inflammatory and oxidative stress biomarkers (C-reactive protein (CRP), tumor necrosis factor (TNF), 4-hydroxynonenal (HNE) and glutathione), metabolic risk markers (blood pressure, waist circumference and glucose, triglyceride and cholesterol levels) and lifestyle habits of the participants (physical activity, smoking and alcohol consumption), a study was carried out using machine learning in a sample of 171 participants, 91 patients with depression (71.42% women, mean age = 50.64) and 80 healthy subjects (67.50% women, mean age = 49.12). The algorithm used was the support vector machine, performing cross validation, by which the subdivision of the sample in training (70%) and test (30%) was carried out in order to estimate the precision of the model. The prediction of belonging to the patient group (MDD patients versus control subjects), melancholic type (melancholic versus non-melancholic patients) or resistant depression group (treatment-resistant versus non-treatment-resistant) was based on the importance of each of the immunometabolic and lifestyle variables. RESULTS: With the application of the algorithm, controls versus patients, such as patients with melancholic symptoms versus non-melancholic symptoms, and resistant versus non-resistant symptoms in the test phase were optimally classified. The variables that showed greater importance, according to the results of the area under the ROC curve, for the discrimination between healthy subjects and patients with depression were current alcohol consumption (AUC = 0.62), TNF-α levels (AUC = 0.61), glutathione redox status (AUC = 0.60) and the performance of both moderate (AUC = 0.59) and vigorous physical exercise (AUC = 0.58). On the other hand, the most important variables for classifying melancholic patients in relation to lifestyle habits were past (AUC = 0.65) and current (AUC = 0.60) tobacco habit, as well as walking routinely (AUC = 0.59) and in relation to immunometabolic markers were the levels of CRP (AUC = 0.62) and glucose (AUC = 0.58). In the analysis of the importance of the variables for the classification of treatment-resistant patients versus non-resistant patients, the systolic blood pressure (SBP) variable was shown to be the most relevant (AUC = 0.67). Other immunometabolic variables were also among the most important such as TNF-α (AUC = 0.65) and waist circumference (AUC = 0.64). In this case, sex (AUC = 0.59) was also relevant along with alcohol (AUC = 0.58) and tobacco (AUC = 0.56) consumption. CONCLUSIONS: The results obtained in our study show that it is possible to predict the diagnosis of depression and its clinical typology from immunometabolic markers and lifestyle habits, using machine learning techniques. The use of this type of methodology could facilitate the identification of patients at risk of presenting depression and could be very useful for managing clinical heterogeneity.

S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells.

BACKGROUND & AIMS: Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NO-mediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CONCLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

Sucrose Hydrolysis in a Continuous Packed-Bed Reactor with Auto-immobilise Aspergillus niger Biocatalyst Obtained by Solid-State Fermentation.

Invertase from Aspergillus niger C28B25 was produced by solid-state fermentation (SSF). Fermented solids were used directly as a biocatalyst for batch and continuous hydrolysis of sucrose in a packed-bed reactor under different operational conditions with various temperatures, sucrose concentrations, and feed flow rates. The SSF allowed obtaining a biocatalyst with an invertase activity of 82.2 U/g db. The biocatalyst maintained its activity in the range of 40 to 70 °C for at least 70 h of continuous operation. In a 20-mL packed bed reactor, the highest hydrolysis rate (12.3 g/g db h) was obtained at 40 °C with 2 M sucrose. Continuous hydrolysis in 20-mL and 200-mL reactors at 60 °C led to sucrose hydrolysis above 60% (8.5 residence times) and above 55% (4.5 residence times), respectively. The auto-immobilised biocatalyst produced by SSF without recovery, purification, and immobilisation stages offers an economical alternative for developing accessible biocatalysts that can be applied in batch or continuous sucrose hydrolysis processes. This study shows the potential of biocatalyst production by SSF for other enzymatic systems.

Relationship between immunometabolic status and cognitive performance among major depression disorder patients.

BACKGROUND: Alterations in cognitive performance have been described in patients with major depressive disorder (MDD). However, the specific risk factors of these changes are not yet known. This study aimed to explore whether inmunometabolic parameters are related to cognitive performance in MDD in comparison to healthy controls (HC) METHODS: Sample consisted of 84 MDD patients and 78 HC. Both groups were compared on the results of cognitive performance measured with the Cambridge Neuropsychological Test Automated Battery (CANTAB), the presence of metabolic syndrome (MetS) and an inflammatory/oxidative index calculated by a principal component analysis of peripheral biomarkers (tumor necrosis factor, C-reactive protein and 4-hydroxynonenal). A multiple linear regression was carried out, to study the relationship between inmunometabolic variables and the global cognitive performance, being the latter the dependent variable. RESULTS: Significant differences were obtained in the inflammatory/oxidative index between both groups (F(1157)= 12.93; p < .001), also in cognitive performance (F(1157)= 56.75; p < .001). The inmunometabolic covariate regression model (i.e., condition (HC/MDD), sex, age and medication loading, MetS, inflammatory/oxidative index and the interaction between MetS and inflammatory/oxidative index) was statistically significant (F(7157)= 11.24; p < .01) and explained 31% of variance. The condition, being either MDD or HD, (B=-0.97; p < .001), age (B=-0.28; p < .001) and the interaction between inflammatory/oxidative index and MetS (B=-0.38; p = .02) were factors associated to cognitive performance. LIMITATIONS: Sample size was relatively small. The cross-sectional design of the study limits the possibilities of analysis. CONCLUSIONS: Our results provide evidence on the conjoint influence of metabolic and inflammatory dysregulation on cognitive dysfunction in MDD patients. In this way, our study opens a line of research in immunometabolic agents to deal with cognitive decline associated with MDD.

Nitric oxide mimics transcriptional and post-translational regulation during α-tocopherol cytoprotection against glycochenodeoxycholate-induced cell death in hepatocytes.

BACKGROUND & AIMS: Reactive oxygen species (ROS) and nitric oxide (NO) exert a relevant role during bile acid-induced hepatotoxicity. Whether α-Tocopherol regulates oxidative and nitrosative stress, bile acid transporter expression and their NO-dependent post-translational modifications, and cell death were assessed in vitro and in vivo. METHODS: α-Tocopherol and/or NO donors (DETA-NONOate or CSNO, and V-PYRRO/NO) were administered to glycochenodeoxycholic acid (GCDCA)-treated cultured human hepatocytes or to bile duct obstructed rats. Cell injury, superoxide anion (O⁻2) production, as well as inducible nitric oxide synthase (NOS-2), cytochrome P4507A1 (CYP7A1), heme oxygenase-1, (HO-1) and bile acid transporter expression were determined. Cysteine S-nitrosylation and tyrosine nitration of Na(+)-taurocholate co-transporting polypeptide (NTCP), as well as taurocholic acid (TC) uptake were also evaluated. RESULTS: GCDCA-induced cell death was associated with increased (O⁻2) production, NTCP and HO-1 expression, and with a reduction of CYP7A1 and NOS-2 expression. α-Tocopherol reduced cell death, (O⁻2) production, CYP7A1, NTCP, and HO-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and tyrosine nitration, and reduced TC uptake in hepatocytes. α-Tocopherol and V-PYRRO/NO reduced liver injury and NTCP expression in obstructed rats. CONCLUSIONS: The regulation of CYP7A1, NTCP, and HO-1 expression may be relevant for the cytoprotective properties of α-Tocopherol and NO against mitochondrial dysfunction, oxidative stress and cell death in GCDCA-treated hepatocytes. The regulation of NO-dependent post-translational modifications of NTCP by α-Tocopherol and NO donors reduces the uptake of toxic bile acids by hepatocytes.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy.

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.