Sodium nitroprusside modulates oxidative and nitrosative processes in Lycopersicum esculentum L. under drought stress.

KEY MESSAGE: Sodium nitroprusside mediates drought stress responses in tomatoes by modulating nitrosative and oxidative pathways, highlighting the interplay between nitric oxide, hydrogen sulfide, and antioxidant systems for enhanced drought tolerance. While nitric oxide (NO), a signalling molecule, enhances plant tolerance to abiotic stresses, its precise contribution to improving tomato tolerance to drought stress (DS) through modulating oxide-nitrosative processes is not yet fully understood. We aimed to examine the interaction of NO and nitrosative signaling, revealing how sodium nitroprusside (SNP) could mitigate the effects of DS on tomatoes. DS-seedlings endured 12% polyethylene glycol (PEG) in a 10% nutrient solution (NS) for 2 days, then transitioned to half-strength NS for 10 days alongside control plants. DS reduced total plant dry weight, chlorophyll a and b, Fv/Fm, leaf water potential (ΨI), and relative water content, but improved hydrogen peroxide (H2O2), proline, and NO content. The SNP reduced the DS-induced H2O2 generation by reducing thiol (-SH) and the carbonyl (-CO) groups. SNP increased not only NO but also the activity of L-cysteine desulfhydrase (L-DES), leading to the generation of H2S. Decreases in S-nitrosoglutathione reductase (GSNOR) and NADPH oxidase (NOX) suggest a potential regulatory mechanism in which S-nitrosylation [formation of S-nitrosothiol (SNO)] may influence protein function and signaling pathways during DS. Moreover, SNP improved ascorbate (AsA) and glutathione (GSH) and reduced oxidized glutathione (GSSG) levels in tomato plants under drought. Furthermore, the interaction of NO and H2S, mediated by L-DES activity, may serve as a vital cross-talk mechanism impacting plant responses to DS. Understanding these signaling interactions is crucial for developing innovative drought-tolerance strategies in crops.

Extended Prophylactic Effect of N-tert-Butyl-α-phenylnitron against Oxidative/Nitrosative Damage Caused by the DNA-Hypomethylating Drug 5-Azacytidine in the Rat Placenta.

Antioxidant N-tert-Butyl-α-phenylnitron (PBN) partly protected embryos from the negative effects of a DNA demethylating drug 5-azacytidine during pregnancy. Our aim was to investigate PBN's impact on the placenta. Fischer rat dams were treated on gestation days (GD) 12 and 13 by PBN (40 mg/kg), followed by 5azaC (5 mg/kg) after one hour. Global methylation was assessed by pyrosequencing. Numerical density was calculated from immunohistochemical expression in single cells for proliferating (PCNA), oxidative (oxoguanosine) and nitrosative (nitrotyrosine) activity. Results were compared with the PBN-treated and control rats. PBN-pretreatment significantly increased placental weight at GD15 and GD20, diminished by 5azaC, and diminished apoptosis in GD 20 placentas caused by 5azaC. Oxoguanosine expression in placentas of 5azaC-treated dams was especially high in the placental labyrinth on GD 15, while PBN-pretreatment lowered its expression on GD 15 and GD 20 in both the labyrinth and basal layer. 5azaC enhanced nitrotyrosine level in the labyrinth of both gestational stages, while PBN-pretreatment lowered it. We conclude that PBN exerted its prophylactic activity against DNA hypomethylating agent 5azaC in the placenta through free radical scavenging, especially in the labyrinthine part of the placenta until the last day of pregnancy.

Novel Tocolytic Strategy: Modulating Cx43 Activity by S-Nitrosation.

Currently available tocolytics are ineffective at significantly delaying preterm birth. This is due in part to our failure to better understand the mechanisms that drive spontaneous preterm labor (sPTL). Cyclic nucleotides are not the primary contributors to myometrial quiescence, but instead nitric oxide (NO)-mediated protein S-nitrosation (SNO) is integral to the relaxation of the tissue. Connexin-43 (Cx43), a myometrial "contractile-associated protein" that functions as either a gap junction channel or an hemichannel (HC), was the focus of this study. Protein analysis determined that Cx43 is downregulated in sPTL myometrium. Furthermore, Cx43 is S-nitrosated by NO, which correlates with an increase of phosphorylated Cx43 at serine 368 (Cx43-pS368 -gap junction inhibition) as well as an increase in the HC open-state probability (quiescence). Pharmacologic inhibition of Cx43 with 18ß-glycyrrhetinic acid (18ß-GA) exhibits a negative inotropic effect on the myometrium in a dose-dependent manner, as does administration of nebivolol, an NO synthase activator that increases total protein SNOs. When 18ß-GA and nebivolol were coadministered at their IC50 values, the effect on contractile dynamics was additive and all but eliminated contractions. The development of new tocolytics demands a better understanding of the underlying mechanisms of sPTL. Here it has been shown that 18ß-GA and nebivolol leverage dysregulated pathways in the myometrium, resulting in a novel approach for the treatment of sPTL. SIGNIFICANCE STATEMENT: Although there are many known causes of preterm labor (PTL), the mechanisms of "spontaneous" PTL (sPTL) remain obfuscated, which is why treating this condition is so challenging. Here we have identified that connexin-43 (Cx43), an important contractile-associated protein, is dysregulated in sPTL myometrium and that the pharmacologic inhibition of Cx43 and its S-nitrosation with 18ß-glycyrrhetinic acid and nebivolol, respectively, significantly blunts contraction in human myometrial tissue, presenting a novel approach to tocolysis that leverages maladjusted pathways in women who experience sPTL.

Sodium nitrite improves hypertension-induced myocardial dysfunction by mechanisms involving cardiac S-nitrosylation.

Although nitrite improves vascular function and lowers blood pressure, its cardiac effects are not completely known. We investigated whether nitrite improves the cardiac function in normotensive and in hypertensive rats. Two-kidney, one-clip hypertension model (2K1C) was induced in Wistar rats. Blood pressure was evaluated by tail-cuff plethysmography over 6 weeks. By the end of week 2, hypertensive and normotensive rats received nitrite (daily dose of 1 or 15 mg/kg) by gavage for 4 weeks. Cardiac morphology and function were performed by transthoracic echocardiography. Intrinsic heart function was evaluated using the isolated heart model (Langendorff's preparation). Starling curves were generated under nitrite (1 µmol/L) and/or ascorbate (1 mmol/L) or vehicle. Cardiac tissue was collected and snap frozen for biochemical analysis. Nitrite treatment (15 mg/kg) lowered both systolic blood pressure and the increases in left ventricular (LV) mass found in 2K1C rats (P < .05). In addition, nitrite treatment restored the decreased cardiac output in 2K1C rats (P < .05) and improved the cardiac function. These findings were associated with increased nitrite, S-nitrosothiols, and protein S-nitrosylation (all P < .05) assessed in heart tissue. The cardiac effects of nitrite were further investigated in the isolated heart model, and nitrite infusion (1 µmol/L) enhanced cardiac contractility and relaxation. This infusion increased S-nitrosothiols concentrations and protein S-nitrosylation in the heart. Ascorbate completely blunted all nitrite-induced effects. These findings show that treatment with oral nitrite improves cardiac function by mechanisms involving increased S-nitrosothiols generation and S-nitrosylation of cardiac proteins. Pharmacological strategies promoting cardiac S-nitrosylation may be useful to improve myocardial function in heart diseases.

Hiding in Plain Sight: Nebivolol Exhibits Compelling Tocolytic Properties.

Preterm birth before 37 weeks of completed gestation results in numerous health consequences for the foetus. Preterm labour leads to preterm birth in over 50% of cases, and no FDA-approved treatment can prevent labour or help a foetus remain in the womb until term. Examination of nitric oxide mediated relaxation signaling in the uterine smooth muscle reveals a role for protein S-nitrosation. The recent discovery of upregulated S-nitrosoglutathione reductase (GSNOR) in spontaneously preterm labouring women has emphasized the need to explore the function of S-nitrosation regulation in the maintenance of uterine quiescence. Here we have examined the ability of nebivolol to relax uterine smooth muscle and tested recent claims that nebivolol is a GSNOR inhibitor. In uterine smooth muscle strips from both mouse and human, nebivolol relaxes oxytocin-induced contractions in a dose dependent manner. Our data indicates that nebivolol has no effect on GSNOR activity, nor does nebivolol inhibit thioredoxin reductase, two of the major protein denitrosylases. The ability of nebivolol to relax uterine smooth muscle is likely the combined effects of increased nitric oxide synthase activity and ß3-adregnegic stimulation.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.

Chrysophanol attenuates nitrosative/oxidative stress injury in a mouse model of focal cerebral ischemia/reperfusion.

Nitrosative/oxidative stress plays an important role in neuronal death following cerebral ischemia/reperfusion (I/R). Chrysophanol (CHR) has been shown to afford significant neuroprotection on ischemic stroke, however, whether its mechanism is related to attenuating nitrosative/oxidative stress is not clear. In the present study, we investigated the effect of CHR on neuronal injury related to nitric oxide (NO) production by using mouse middle cerebral artery occlusion (MCAO) model. Our results revealed that nitrite plus nitrate (NOx-) and 3-nitrotyrosine (3-NT) levels increased in ischemic brain 14 days after reperfusion, and were subsequently attenuated by CHR treatment. Moreover, 3-NT is colocalized with NeuN and TUNEL, suggesting that neuronal apoptosis following I/R is associated with 3-NT and CHR suppresses NO-associated neuronal cell death. Accordingly, cleaved caspase-3 expression in ischemic brain was decreased by CHR treatment. I/R also decreased the activity of total superoxide dismutase (SOD) and manganese-dependent SOD (MnSOD), whilst increased reactive oxygen species (ROS) production significantly. Interestingly, CHR reversed this decrease in total SOD, and MnSOD activity, and inhibited ROS generation in the ischemic brain. Taken together, our results provide direct evidence suggesting that CHR attenuates nitrosative/oxidative stress injury induced by I/R, providing a novel therapeutic target in the treatment of acute ischemic stroke.

Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells.

Plant-derived pentacyclic triterpenotids with multiple biological activities are considered as promising candidates for cancer therapy and prevention. However, their mechanisms of action are not fully understood. In the present study, we have analyzed the effects of low dose treatment (5-20 µM) of ursolic acid (UA) and betulinic acid (BA) on breast cancer cells of different receptor status, namely MCF-7 (ER+, PR+/-, HER2-), MDA-MB-231 (ER-, PR-, HER2-) and SK-BR-3 (ER-, PR-, HER2+). UA-mediated response was more potent than BA-mediated response. Triterpenotids (5-10 µM) caused G0/G1 cell cycle arrest, an increase in p21 levels and SA-beta-galactosidase staining that was accompanied by oxidative stress and DNA damage. UA (20 µM) also diminished AKT signaling that affected glycolysis as judged by decreased levels of HK2, PKM2, ATP and lactate. UA-induced energy stress activated AMPK that resulted in cytotoxic autophagy and apoptosis. UA-mediated elevation in nitric oxide levels and ATM activation may also account for AMPK activation-mediated cytotoxic response. Moreover, UA-promoted apoptosis was associated with decreased pERK1/2 signals and the depolarization of mitochondrial membrane potential. Taken together, we have shown for the first time that UA at low micromolar range may promote its anticancer action by targeting glycolysis in phenotypically distinct breast cancer cells.

MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1.

Endothelial dysfunction played an important role in the progression of diabetes mellitus (DM). miR-181c has been implicated in many diseases, including DM. However, the molecular mechanisms of miR-181c regulate this process remained poorly understood. Healthy ICR mice were divided into control group (n = 10) and db/db DM group (n = 10). The expression of miR-181c and FoxO1 were both investigated in diabetic db/db mice or high glucose-induced endothelial cells (MAECs and END-D). Here we found that down-regulation of miR-181c and the activation of FoxO1/iNOS were observed in mice and endothelial cells. Furthermore, we verified that miR-181c directly targeted and inhibited FoxO1 gene expression by targeting its 3'-UTR through luciferase reporter assay. Knockdown of FoxO1 reversed the up-regulation of iNOS, nitrotyrosine and the down-regulation of p-eNOSSer1177/eNOS in high glucose (30 mM)-induced MAECs cells. In addition, over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-FoxO1 in MAECs. These results shown that miR-181c attenuated nitration stress through regulating FoxO1 expression and affecting endothelial cell function, which offering a new target for the development of preventive or therapeutic agents against DM.

Sodium Phenylbutyrate and Edaravone Abrogate Chronic Restraint Stress-Induced Behavioral Deficits: Implication of Oxido-Nitrosative, Endoplasmic Reticulum Stress Cascade, and Neuroinflammation.

Chronic stress exposure can produce deleterious effects on the hippocampus (HC) which eventually leads to cognitive impairment and depression. Endoplasmic reticulum (ER) stress has been reported as one of the major culprits in the development of stress-induced cognitive impairment and depression. We investigated the neuroprotective efficacy of sodium phenylbutyrate (SPB), an ER stress inhibitor, and edaravone, a free radical scavenger, against chronic restraint stress (CRS)-induced cognitive deficits and anxiety- and depressive-like behavior in mice. Adult male Swiss albino mice were restrained for 6 h/day for 28 days and injected (i.p.) with SPB (40 and 120 mg/kg) or edaravone (3 and 10 mg/kg) for the last seven days. After stress cessation, the anxiety- and depressive-like behavior along with spatial learning and memory were examined. Furthermore, oxido-nitrosative stress, proinflammatory cytokines, and gene expression level of ER stress-related genes were assessed in HC and prefrontal cortex (PFC). CRS-exposed mice showed anxiety- and depressive-like behavior, which was significantly improved by SPB and edaravone treatment. In addition, SPB and edaravone treatment significantly alleviated CRS-induced spatial learning and memory impairment. Furthermore, CRS-evoked oxido-nitrosative stress, neuroinflammation, and depletion of Brain-derived neurotrophic factor were significantly ameliorated by SPB and edaravone treatment. We found significant up-regulation of ER stress-related genes in both HC and PFC regions, which were suppressed by SPB and edaravone treatment in CRS mice. Our study provides evidence that SPB and edaravone exerted neuroprotective effects on CRS-induced cognitive deficits and anxiety- and depressive-like behavior, which is possibly coupled with inhibition of oxido-nitrosative stress, neuroinflammation, and ER stress cascade.

Vitamin E mitigates cisplatin-induced nephrotoxicity due to reversal of oxidative/nitrosative stress, suppression of inflammation and reduction of total renal platinum accumulation.

Cisplatin (CP) is one of the most effective chemotherapeutic agents. Unfortunately, CP-induced nephrotoxicity hampered its use. This study aims to investigate the effect of vitamin E (Vit E) on CP-induced nephrotoxicity. Male white albino rats were divided to four group's six rats each and received either, 1% tween 80 in normal saline or Vit E (75 mg/kg) per day for 14 consecutive days or a single injection of CP (6 mg/kg) alone or CP (6 mg/kg) together with Vit E (75 mg/kg per day for 14 consecutive days). Five days after the CP injection, rats were euthanized; blood samples were collected; kidneys were dissected; and biochemical, immunohistochemical, and histological examinations were performed. Our results revealed that CP treatment significantly increased serum levels of creatinine and urea. Moreover, reduced glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities were significantly reduced with concurrent increase in kidney malondialdehyde (MDA) content following CP treatment. Vit E successfully lowered serum levels of urea and creatinine, enhanced creatinine clearance and diuresis, and normalized relative kidney/body weight. Furthermore, Vit E successfully normalized renal MDA and nitrite concentrations, elevated GSH level, and restored CAT and SOD activities in renal tissues. Histopathological examination of rat kidney revealed that Vit E significantly mitigated CP-induced renal damage. Importantly, administration of Vit E reduced kidney total platinum concentration indicating a role of platinum renal accumulation on the ability of Vit E to protect against CP nephrotoxicity.

Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal.

Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells.

Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm.

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

H2S Attenuates LPS-Induced Acute Lung Injury by Reducing Oxidative/Nitrative Stress and Inflammation.

BACKGROUND: Hydrogen sulfide (H2S), known as the third endogenous gaseous transmitter, has received increasing attention because of its diverse effects, including angiogenesis, vascular relaxation and myocardial protection.We aimed to investigate the role of H2S in oxidative/nitrative stress and inflammation in acute lung injury (ALI) induced by endotoxemia. METHODS: Male ICR mice were divided in six groups: (1) Control group; (2) GYY4137treatment group; (3) L-NAME treatment group; (4) lipopolysaccharide (LPS) treatment group; (5) LPS with GYY4137 treatment group; and (6) LPS with L-NAME treatment group. The lungs were analysed by histology, NO production in the mouse lungs determined by modified Griess (Sigma-Aldrich) reaction, cytokine levels utilizing commercialkits, and protein abundance by Western blotting. RESULTS: GYY4137, a slowly-releasing H2S donor, improved the histopathological changes in the lungs of endotoxemic mice. Treatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, increased anti-oxidant biomarkers such as thetotal antioxidant capacity (T-AOC) and theactivities of catalase (CAT) and superoxide dismutase (SOD) but decreased a marker of peroxynitrite (ONOO-) action and 3-nitrotyrosine (3-NT) in endotoxemic lung. L-NAME administration also suppressed inflammation in endotoxemic lung, as evidenced by the decreased pulmonary levels of interleukin (IL)-6, IL-8, and myeloperoxidase (MPO) and the increased level of anti-inflammatory cytokine IL-10. GYY4137 treatment reversed endotoxin-induced oxidative/nitrative stress, as evidenced by a decrease in malondialdehyde (MDA), hydrogenperoxide (H2O2) and 3-NT and an increase in the antioxidant biomarker ratio of reduced/oxidized glutathione(GSH/GSSG ratio) and T-AOC, CAT and SOD activity. GYY4137 also attenuated endotoxin-induced lung inflammation. Moreover, treatment with GYY4137 inhibited inducible NOS (iNOS) expression and nitric oxide (NO) production in the endotoxemia lung. CONCLUSIONS: GYY4137 conferred protection against acute endotoxemia-associated lung injury, which may have beendue to the anti-oxidant, anti-nitrative and anti-inflammatory properties of GYY4137. The present findings warrant further exploration of the clinical applicability of H2S in the prevention and treatment of ALI.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis.

Morin Hydrate Mitigates Cisplatin-Induced Renal and Hepatic Injury by Impeding Oxidative/Nitrosative Stress and Inflammation in Mice.

Cisplatin is a widely used chemotherapeutic drug; however, it induces damage on kidney and liver at clinically effective higher doses. Morin hydrate possesses antioxidant, anti-inflammatory, and anticancer properties. Therefore, we aimed to investigate the effects of morin hydrate (50 and 100 mg/kg, orally) against the renohepatic toxicity induced by a high dose of cisplatin (20 mg/kg, intraperitoneally). Renal and hepatic function, oxidative/nitrosative stress, and inflammatory markers along with histopathology were evaluated. Morin hydrate ameliorated cisplatin-induced renohepatic toxicity significantly at 100 mg/kg as evidenced from the significant reversal of cisplatin-induced body weight loss, mortality, functional and structural alterations of kidney, and liver. The protective role offered by morin hydrate against cisplatin-induced renohepatic toxicity is by virtue of its free radical scavenging property, thereby abating the depletion of cellular antioxidant defense components and through modulation of inflammatory cytokines. We speculate morin hydrate as a protective candidate against renohepatic toxicity of cisplatin.

The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress.

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.

Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells.

BACKGROUND: Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophil-related cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug. OBJECTIVES: We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS-2B ± PMNs stimulated with LPS (1 µg/ml). METHODS: The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test. RESULTS: In BEAS-2B alone, LPS (at 12 h) increased RANTES/iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release (p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS-2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL-8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro. CONCLUSION: The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections.

Nitric oxide mediates selective degeneration of hypothalamic orexin neurons through dysfunction of protein disulfide isomerase.

We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

Effect of methionine sulfoxide reductase B1 (SelR) gene silencing on peroxynitrite-induced F-actin disruption in human lens epithelial cells.

F-actin plays a crucial role in fundamental cellular processes, and is extremely susceptible to peroxynitrite attack due to the high abundance of tyrosine in the peptide. Methionine sulfoxide reductase (Msr) B1 is a selenium-dependent enzyme (selenoprotein R) that may act as a reactive oxygen species (ROS) scavenger. However, its function in coping with reactive nitrogen species (RNS)-mediated stress and the physiological significance remain unclear. Thus, the present study was conducted to elucidate the role and mechanism of MsrB1 in protecting human lens epithelial (hLE) cells against peroxynitrite-induced F-actin disruption. While exposure to high concentrations of peroxynitrite and gene silencing of MsrB1 by siRNA alone caused disassembly of F-actin via inactivation of extracellular signal-regulated kinase (ERK) in hLE cells, the latter substantially aggravated the disassembly of F-actin triggered by the former. This aggravation concurred with elevated nitration of F-actin and inactivation of ERK compared with that induced by the peroxynitrite treatment alone. In conclusion, MsrB1 protected hLE cells against the peroxynitrite-induced F-actin disruption, and the protection was mediated by inhibiting the resultant nitration of F-actin and inactivation of ERKs.