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Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD + as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S -nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO - ) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO - (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO - can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.

Peripheral neurotoxicity is a dose-limiting adverse reaction of primary frontline chemotherapeutic agents, including vincristine. Neuropathy can be so disabling that patients drop out of potentially curative therapy, negatively impacting cancer prognosis. The hallmark of vincristine neurotoxicity is axonopathy, yet its underpinning mechanisms remain uncertain. We developed a comprehensive drug discovery platform to identify neuroprotective agents against vincristine-induced neurotoxicity. Among the hits identified, SIN-1-an active metabolite of molsidomine-prevents vincristine-induced axonopathy in both motor and sensory neurons without compromising vincristine anticancer efficacy. Mechanistically, we found that SIN-1's neuroprotective effect is mediated by activating soluble guanylyl cyclase. We modeled vincristine-induced peripheral neurotoxicity in rats to determine molsidomine therapeutic potential in vivo. Vincristine administration induced severe nerve damage and mechanical hypersensitivity that were attenuated by concomitant treatment with molsidomine. This study provides evidence of the neuroprotective properties of molsidomine and warrants further investigations of this drug as a therapy for vincristine-induced peripheral neurotoxicity., (© 2024. The Author(s).)

The cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by nitric oxide (NO). During the early developmental period, rat hearts exhibit higher resistance to ischemia-reperfusion (I/R) injury, contain higher levels of serum nitrates, and their resistance cannot be further increased by IPC or IPoC. NOS blocker (L-NAME) lowers their high resistance. Wistar rat hearts (postnatal Days 1 and 10) were perfused according to Langendorff and exposed to 40 min of global ischemia followed by reperfusion with or without IPoC. NO and reactive oxygen species donors (DEA-NONO, SIN-1) and L-NAME were administered. Tolerance to ischemia decreased between Days 1 and 10. DEA-NONO (low concentrations) significantly increased tolerance to I/R injury on both Days 1 and 10. SIN-1 increased tolerance to I/R injury on Day 10, but not on Day 1. L-NAME significantly reduced resistance to I/R injury on Day 1, but actually increased resistance to I/R injury on Day 10. Cardioprotection by IPoC on Day 10 was not affected by either NO donors or L-NAME. It can be concluded that resistance of the neonatal heart to I/R injury is NO dependent, but unlike in adult hearts, cardioprotective interventions, such as IPoC, are most likely NO independent., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Numerous research projects focused on the management of acute pulmonary hypertension as Coronavirus Disease 2019 (COVID-19) might lead to hypoxia-induced pulmonary vasoconstriction related to acute respiratory distress syndrome. For that reason, inhalative therapeutic options have been the subject of several clinical trials. In this experimental study, we aimed to examine the hemodynamic impact of the inhalation of the SIN-1A formulation (N-nitroso-N-morpholino-amino-acetonitrile, the unstable active metabolite of molsidomine, stabilized by a cyclodextrin derivative) in a porcine model of acute pulmonary hypertension. Landrace pigs were divided into the following experimental groups: iNO (inhaled nitric oxide, n = 3), SIN-1A-5 (5 mg, n = 3), and SIN-1A-10 (10 mg, n = 3). Parallel insertion of a PiCCO system and a pulmonary artery catheter (Swan-Ganz) was performed for continuous hemodynamic monitoring. The impact of iNO (15 min) and SIN-1A inhalation (30 min) was investigated under physiologic conditions and U46619-induced acute pulmonary hypertension. Mean pulmonary arterial pressure (PAP) was reduced transiently by both substances. SIN-1A-10 had a comparable impact compared to iNO after U46619-induced pulmonary hypertension. PAP and PVR decreased significantly (changes in PAP: -30.1% iNO, -22.1% SIN-1A-5, -31.2% SIN-1A-10). While iNO therapy did not alter the mean arterial pressure (MAP) and systemic vascular resistance (SVR), SIN-1A administration resulted in decreased MAP and SVR values. Consequently, the PVR/SVR ratio was markedly reduced in the iNO group, while SIN-1A did not alter this parameter. The pulmonary vasodilatory impact of inhaled SIN-1A was shown to be dose-dependent. A larger dose of SIN-1A (10 mg) resulted in decreased PAP and PVR in a similar manner to the gold standard iNO therapy. Inhalation of the nebulized solution of the new SIN-1A formulation (stabilized by a cyclodextrin derivative) might be a valuable, effective option where iNO therapy is not available due to dosing difficulties or availability.

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency. NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Background: Previous studies suggested that Cg1 area of the cingulate cortex of rats controls glutamate-mediated fear-induced defensive behaviour and antinociception organised at the posterior hypothalamus. In turn, microinjection of the nitric oxide donor SIN-1 into the anterior hypothalamus of mice produced defensive behaviours and fear-induced antinociception. However, it remains unknown whether Cg1 also modulates the latter mechanisms in mice., Aims: The present study examined the influence of Cg1 on SIN1-evoked fear-induced defensive behaviour and antinociception organised at the anterior hypothalamus of mice., Methods: The fear-like behavioural and antinociceptive responses to the microinjection of SIN-1 (300 nmol) into the anterior hypothalamus were evaluated after the microinjection of either N-methyl-D-aspartic acid receptor agonist (0.1, 1 and 10 nmol) or physiological saline into the cingulate cortex of C57BL/6 male mice. In addition, neurotracing and immunohistochemistry were used to characterise Cg1-anterior hypothalamus glutamatergic pathways., Results: The data showed that activation of Cg1 N-methyl-D-aspartic acid receptors increased escape while reducing freezing and antinociceptive responses to SIN-1 microinjections into the anterior hypothalamus. Anterograde neural tract tracer co-localised with VGLUT2-labelled fibres suggests these responses are mediated by glutamatergic synapses at the anterior hypothalamus., Conclusions: In contrast with previous studies showing that Cg1 facilitates both escape and antinociception to chemical stimulation of the posterior hypothalamus in rats, the present data suggest that Cg1 facilitates escape while inhibiting defensive antinociception produced by the microinjection of SIN-1 in the anterior hypothalamus of mice. Accordingly, Cg1 may have opposite effects on antinociceptive responses organised in the anterior and posterior hypothalamus of mice and rats, respectively.

Molsidomine is currently used as a vasodilator drug for the treatment of myocardial ischemic syndrome and congestive heart failure, although still presenting some mitochondrial-targeted side effects in many human cells. As a model of molsidomine mitotoxicity, the reaction of cytochrome c with phosphatidylserine (PS)- and cardiolipin (CL)-containing liposomes was investigated in oxidative/nitrosative conditions imposed by SIN-1 decomposition, which renders peroxynitrite (ONOO - ) as a main reactive product. In these conditions, the production of thiobarbituric acid-reactive substance (TBARs) and LOOH was affected by the lipid composition and the oxidative/nitrative conditions used. The oxidative/nitrative conditions were the exposure of lipids to SIN-1 decomposition, native cytochrome c after previous exposure to SIN-1, concomitantly to SIN-1 and native cytochrome c, native cytochrome c, and cytochrome c modified by SIN-1 that presents a less-rhombic heme iron (L-R cytc). TBARs and LOOH production by lipids and cytochrome c exposed concomitantly to SIN-1 differed from that obtained using L-R cytc and featured similar effects of SIN-1 alone. This result suggests that lipids rather than cytochrome c are the main targets for oxidation and nitration during SIN-1 decomposition. PS- and CL-containing liposomes challenged by SIN-1 were analyzed by Fourier transform infrared spectroscopy that revealed oxidation, trans-isomerization, and nitration. These products are consistent with reaction routes involving lipids and NOx formed via peroxynitrite or direct reaction of NO • with molecular oxygen that attacks LOOH and leads to the formation of substances that are not reactive with thiobarbituric acid.

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu 2+ ), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH ( P < 0.05; IC 50 PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18  μ g/ml) and peroxynitrite scavenging assays. PIT at 75  μ g/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu 2+ , SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST ( P < 0.05; IC 50 PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67  μ g/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Kittipot Sirichaiwetchakoon et al.)

Aim: Brain nitric oxide (NO) have been reported in regulation of the sympatho-adrenomedullary system, which can affect voiding and storage functions. Therefore, we investigated effects of intracerebroventricularly (icv) administered 3-(4-morpholinyl)sydnonimine, hydrochloride (SIN-1) (NO donor) on the micturition reflex, focusing on their dependence on the sympatho-adrenomedullary system and on brain N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats., Methods: Plasma noradrenaline and adrenaline were measured just before and 5 minutes after SIN-1 administration. Evaluation of urodynamic parameters was started 1 hour before SIN-1 administration or intracerebroventricular pretreatment with other drugs., Results: SIN-1 (100 and 250 µg/animal) elevated plasma adrenaline and reduced intercontraction interval ([ICI] values; 110.5% [SIN-1, 0 µg] and 54.9% [SIN-1, 250 µg] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.59]) without affecting plasma noradrenaline or maximal voiding pressure. SIN-1 (250 µg/animal) reduced single-voided volume and bladder capacity without affecting post-voiding residual volume. The SIN-1 (250 µg/animal)-induced adrenaline elevation and ICI reduction were attenuated by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) (NO scavenger, icv) (ICI values; 44.7% [vehicle + SIN-1] and 77.5% [carboxy-PTIO + SIN-1] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.51]). Acute bilateral adrenalectomy abolished SIN-1-induced adrenaline elevation, while showed no effect on the SIN-1-induced ICI reduction. The ICI reduction was attenuated by MK-801 (NMDA receptor antagonist, icv) (ICI values; 47.0% [vehicle + SIN-1] and 87.6% [MK-801 + SIN-1] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.61]), but not by DNQX (AMPA receptor antagonist, icv)., Conclusion: Brain NO is involved in facilitation of the rat micturition reflex through brain NMDA receptors, independently of the sympatho-adrenomedullary outflow modulation., (© 2020 Wiley Periodicals LLC.)

We recently showed that red blood cells (RBCs) from patients with type 2 diabetes mellitus (T2DM-RBCs) induce endothelial dysfunction through a mechanism involving arginase I and reactive oxygen species. Peroxynitrite is known to activate arginase in endothelial cells. Whether peroxynitrite regulates arginase activity in RBCs, and whether it is involved in the cross-talk between RBCs and the vasculature in T2DM, is unclear and elusive. The present study was designed to test the hypothesis that endothelial dysfunction induced by T2DM-RBCs is driven by peroxynitrite and upregulation of arginase. RBCs were isolated from patients with T2DM and healthy age matched controls. RBCs were co-incubated with aortae isolated from wild type rats for 18 h in the absence and presence of peroxynitrite scavenger FeTTPS. Evaluation of endothelial function in organ chambers by cumulative addition of acetylcholine as well as measurement of RBC and vessel arginase activity was performed. In another set of experiments, RBCs isolated from healthy subjects (Healthy RBCs) were incubated with the peroxynitrite donor SIN-1 with subsequent evaluation of endothelial function and arginase activity. T2DM-RBCs, but not Healthy RBCs, induced impairment in endothelial function, which was fully reversed by scavenging of RBC but not vascular peroxynitrite with FeTPPS. Arginase activity was up-regulated by the peroxynitrite donor SIN-1 in Healthy RBCs, an effect that was inhibited by FeTTPS. Healthy RBCs co-incubated with aortae in the presence of SIN-1 caused impairment of endothelial function, which was inhibited by FeTTPS or the arginase inhibitor ABH. T2DM-RBCs induced up-regulation of vascular arginase, an effect that was fully inhibited by FeTTPS. Collectively, our data indicate that RBCs impair endothelial function in T2DM via an effect that is driven by a peroxynitrite-mediated increase in arginase activity. This mechanism may be targeted in patients with T2DM for improvement in endothelial function.

Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate-glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine-cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate-glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO - ) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO - donor (SIN-1) or directly with ONOO - , which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO - treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate ( g app ) in combination with a conservation of the force redevelopment rate (k Tr ) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

Several studies have shown that peroxynitrite (ONOO-), formed upon the reaction of •NO and O2-, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO--induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO- donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1-200 μM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO- donor with FeTMPyP (1 μM), an ONOO- decomposition catalyst, inhibited the effects induced by ONOO-. Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO- (SIN-1 200 μM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO- induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO-. Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO- in cardiac tissue.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

The ability to detect and image secreted peroxynitrite (ONOO - ) along the extracellular surface of a single cell is biologically significant, as ONOO - generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO - , precise determination of the ONOO - level at the cell surface remains a challenging task. In this paper, the use of a membrane-anchored streptavidin-biotin-controlled binding probe (CBP), ONOO-CBP, to determine quantitatively the ONOO - level at the cell surface and to investigate the effect of different stimulants on the production of ONOO - along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO - secretion, achieving more than a 25-fold increase in ONOO - relative to untreated cells. After 1 h of phorbol-12-myristate-13-acetate (PMA) stimulation, the amount of ONOO - secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3-morpholinosydnonimine hydrochloride (SIN-1), which was estimated to release about 20 μm of ONOO - into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We show that 3-morpholinosydnonimine (SIN-1)-induced nitric oxide (NO) triggers the formation of SGs. Whereas the composition of NO-induced SGs is initially similar to sodium arsenite (SA)-induced type I (cytoprotective) SGs, the progressive loss of eIF3 over time converts them into pro-death (type II) SGs. NO-induced SG assembly requires the phosphorylation of eIF2α, but the transition to type II SGs is temporally linked to the mTOR-regulated displacement of eIF4F complexes from the m 7 guanine cap. Whereas SA does not affect mitochondrial morphology or function, NO alters mitochondrial integrity and function, resulting in increased ROS production, decreased cytoplasmic ATP, and plasma membrane permeabilization, all of which are supported by type II SG assembly. Thus, cellular energy balance is linked to the composition and function of NO-induced SGs in ways that determine whether cells live or die.

Peroxynitrite-mediated nitrosative stress in the brain has been associated with various neurodegenerative disorders. Recent evidence highlights peroxisome proliferator-activated receptor γ (PPAR γ ) as a critical neuroprotective factor in neurodegenerative diseases. Here, we observed the effect of the herb hydroxysafflor yellow A (HSYA) during nitrosative stress in neurons and investigated the mechanism based on PPAR γ protection. We found that a single exposure of primary neurons to peroxynitrite donor SIN-1 caused neuronal injury, which was accompanied by the increase of PPAR γ nitration status and lack of activation of the receptor, as measured by PPAR γ DNA-binding activity, by agonist (15d-PGJ2 or rosiglitazone) stimulation. The crucial role of PPAR γ in neuronal defense against nitrosative stress was verified by showing that pretreatment with 15d-PGJ2 or rosiglitazone attenuated SIN-1-induced neuronal injury but pretreatment with GW9662, a PPAR γ antagonist, aggravated SIN-1-induced neuronal injury. The addition of HSYA not only inhibited SIN-1-induced neuronal damage but prevented PPAR γ nitrative modification and resumed PPAR γ activity stimulated by either 15d-PGJ2 or rosiglitazone. Furthermore, HSYA also showed the ability to rescue the neuroprotective effect of 15d-PGJ2 or rosiglitazone when the agonists were coincubated with SIN-1. Finally, in vivo experiments demonstrated that the administration of HSYA also efficiently blocked PPAR γ nitration and loss of activity in the SIN-1-injected hippocampus and reversed the increased neuronal susceptibility which was supported by the inhibition of Bcl-2 protein downregulation induced by SIN-1. The results suggest that HSYA protects neurons from nitrosative stress through keeping PPAR γ as a functional receptor, allowing a more effective activation of this neuroprotective factor by the endogenous or exogenous agonist. Our findings provide new clues in understanding the role of the neuroprotective potential of the herbal HSYA.

Chondrocyte is targeted for disruption in Osteoarthritis (OA) and Kashin-Beck Disease (KBD), and chondrocyte death in cartilage may contribute to the progression of OA and KBD. Oxidative stress leads to increased risk for OA. Previous work in our laboratory implicates oxidative stress as a potential mediator in children with KBD. While these studies suggest a role for oxidative stress in the modulation of OA and KBD, the direct effects of reactive oxygen species/reactive nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, decreased the cell viability in hypertrophic chondrocytes in a dose- and time- dependent manner. SIN-1 induced necrosis in hypertrophic chondrocytes, whereas triggered apoptosis in non-hypertrophic cells of non-differentiated ATDC5 cells and C28/I2 cells. Ultrastructural analysis of hypertrophic chondrocyte treated with SIN-1 revealed morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and organelles, even to disappearance. Moreover, SIN-1 induced chondronecrosis in the deep zone of engineered cartilage tissue, such as cell-free vacancy and "red ghost" cells. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenous ROS/RNS, leads to necrosis in hypertrophic chondrocytes. Oxidative stress-mediated necrotic cell death contributes to chondronecrosis in the deep zone of cartilage in both OA and KBD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Objective: The aim of this study was to investigate FGF8 and FGFR3 expression in clinical samples of Kashin-Beck disease (KBD), an endemic osteochondropathy found in China, as well as in pre-clinical models of this disease., Method: Cartilage was collected from the hand phalanges of five patients with KBD and from five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for four weeks prior to exposure to the T-2 toxin. ATDC5 cells were differentiated into hypertrophic chondrocytes for twenty-one days, and then treated with 3-morpholinosydnonimine (SIN-1) (0, 1, 3, or 5 mM) for 24 h. FGF8 and FGFR3 were visualized using immunohistochemistry; protein levels were assessed by western blotting, and mRNA levels were determined by real-time RT-PCR., Results: Increased staining of FGF8 and FGFR3 was observed in the cartilage of children with KBD compared to normal children. Both increased FGF8 and FGFR3 staining, as well as protein levels, were also observed in the cartilage of rats fed normal or Se-deficient diets plus T-2 toxin exposure, compared to those in rats fed with normal or Se-deficient diets alone. SIN-1 treatment of hypertrophic chondrocytes (ATCD5 cells) increased FGF8 and FGFR3 protein and mRNA levels in a dose-dependent manner., Conclusion: Our data indicate that SIN-1 induces FGF8 and FGFR3 overexpression and this is involved in the abnormal terminal differentiation and degradation of the ECM in cartilage. FGF8 and FGFR3 may therefore play an important role in the onset of deep zone necrosis and pathogenesis in KBD in adolescent children., (Copyright © 2018. Published by Elsevier Inc.)

Background: An excitatory imbalance in the hypothalamus of rodents caused by local chemical stimulation elicits fear-related defensive reactions such as escape and freezing. In addition, these panic attack-like defensive reactions induced by hypothalamic neurons may cause antinociception. However, there is a shortage of studies showing the participation of the anterior hypothalamic nucleus in these adaptive defensive mechanisms. Nitric oxide (NO) donors have been shown to evoke fear-related defensive responses when microinjected into paralimbic and limbic structures, and this excitatory neuromodulation can recruit the glutamatergic system., Aims: The aim of this work was to investigate the influence of the glutamatergic system in the nitrergic effects on fear-related defensive responses organised by anterior hypothalamic neurons., Methods: The present study evaluates the effects of the molsidomine active metabolite SIN-1 NO donor administered into the anterior hypothalamus (AH) of mice at different concentrations (75, 150 and 300 nmol/0.1 μL). Then, we investigated the effects of pre-treatment of the AH with AP-7 (an N-methyl-d-aspartate (NMDA) receptor-selective antagonist; 0.02, 0.2 and 2 nmol/0.1 μL) on the behavioural and antinociceptive effects provoked by AH chemical stimulation with SIN-1 microinjections., Results: The 300 nmol dose of SIN-1 was the most effective at causing panic-like defensive behaviours followed by a significant antinociceptive response. In addition, both of these effects were attenuated or inhibited by AH pre-treatment with AP-7., Conclusions: These findings suggest that the panicogenic and antinociceptive effects evoked by intra-AH microinjections of SIN-1 depend on NMDA receptor activation.

The aim of this study was to assess whether there are differences between the results of determining oxidative stress markers obtained from different origin cell lines after exposure to chemicals generating free radicals. The studies considered two markers of oxidative stress: the level of thiobarbituric acid reactive substances (TBARS) and superoxide dismutase activity. The evaluation was performed in five cell lines: Chinese hamster ovary (CHO-9) cells, lung adenocarcinoma A549, macrophages RAW264.7, skin carcinoma cells A431, and keratinocytes HaCaT. Three compounds generating free radicals were used as a source of reactive oxygen/nitrogen: 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), sodium persulfate (SP), and 3-morpholinosydnonimine hydrochloride (SIN-1). The most appropriate cell line to assess the level of TBARS proved to be the murine macrophage cell line RAW 264.7. Equally, good performance was observed in the lung cancer cell line A549, but only when tested with AAPH and SP. In the case of measuring superoxide dismutase activity, it appeared that the most suitable cell line was also the RAW 264.7 line, although dispersion increased significantly at the highest concentrations of AAPH and SP measurements. When choosing a cell line to determine oxidative stress, the specificity of the stress-inducing compound and the parameter determined should be taken into consideration.

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.

The axon initial segment (AIS), the domain responsible for action potential initiation and maintenance of neuronal polarity, is targeted for disruption in a variety of central nervous system pathological insults. Previous work in our laboratory implicates oxidative stress as a potential mediator of structural AIS alterations in two separate mouse models of central nervous system inflammation, as these effects were attenuated following reactive oxygen species scavenging and NADPH oxidase-2 ablation. While these studies suggest a role for oxidative stress in modulation of the AIS, the direct effects of reactive oxygen and nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, drives a reversible loss of AIS protein clustering in primary cortical neurons in vitro. Pharmacological inhibition of both voltage-dependent and intracellular calcium (Ca 2+ ) channels suggests that this mechanism of AIS disruption involves Ca 2+ entry specifically through L-type voltage-dependent Ca 2+ channels and its release from IP 3 -gated intracellular stores. Furthermore, ROS/RNS-induced AIS disruption is dependent upon activation of calpain, a Ca 2+ -activated protease previously shown to drive AIS modulation. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenously applied ROS/RNS, is capable of driving structural alterations in the AIS complex.

Peroxynitrite and heme peroxidases (or heme)-H 2 O 2 -NaNO 2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H 2 O 2 -NaNO 2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H 2 O 2 -NaNO 2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H 2 O 2 -NaNO 2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H 2 O 2 -NaNO 2 -induced nitration. Our results indicated that peroxynitrite and heme-H 2 O 2 -NaNO 2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening., (© 2017 Wiley Periodicals, Inc.)

Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS., (© 2016 Blackwell Verlag GmbH.)

Molsidomine is a well-known vasodilatating, antianginal drug. Despite earlier studies with its metabolites (3-morpholino-syndnonimine (SIN-1) and N-nitroso-N-morpholino-amino-acetonitrile (SIN-1A)), which indicated a potential favorable cardioprotective activity, a lot of controversy remains. The aim of our research was to compare molsidomine, SIN-1, SIN-1A, and lidocaine influence on arrhythmias and hemodynamic parameters in 2 experimental models in rats. In the Langendorff heart study, SIN-1A markedly elevated left ventricular systolic pressure, maximum rise and fall of the first pressure derivative, coronary flow, and myocardial oxygen consumption. In addition, SIN-1A more so than SIN-1 significantly lowered creatine kinase release. The antiarrhythmic action of SIN-1 was observed, while lidocaine significantly diminished ventricular arrhythmias duration in comparison with the control. In the ischemia-reperfusion-induced arrhythmias model, hypotensive action of molsidomine was observed as well as the reduction in pressure rate product. Molsidomine also prolonged ventricular tachycardia duration. On the other hand, no significant effects on hemodynamic parameters as well as on ventricular arrhythmias were found in any of the SIN-1 and SIN-1A groups. In conclusion, our research suggests a possible direct, cardioprotective action of SIN-1A. It seems worthwhile to further investigate molsidomine derivatives, especially SIN-1A, because of its potential use in invasive cardiology procedures such as percutaneous transluminal coronary angioplasty.

Background: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking., Objective: By using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format., Methods: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes., Results: Beer's law was linear over a nanomolar range (1-10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10-1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, N ω -Nitro-l-arginine methyl ester (l-NAME)-the standard inhibitor of endothelial NO synthase-was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh., Conclusions: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability.

Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100 μM SIN-1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production., (© 2016 Blackwell Verlag GmbH.)

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a-3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility., Abbreviations: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation, Anova: analysis of variance.

Nitrous oxide (N2O)-induced antinociception in mice is dependent on the neuromodulator nitric oxide (NO). In contrast to C57BL/6J (B6) mice, DBA/2J (D2) mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of D2 mice to respond to N2O might result from a deficit of NO function. Therefore, it was of interest to determine whether increasing the availability of NO might increase sensitivity of D2 mice to N2O. Male D2 mice were pretreated with sub-antinociceptive intracerebroventricular doses of the NO donor 3-morpholinosydnoimine or the NO precursor l-arginine then assessed for responsiveness to N2O-induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of D2 mice to N2O. These results indicate that the NOS enzyme in D2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O-induced antinociception may lie in availability or utilization of l-arginine., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

Background: Nitric oxide (NO) can reduce platelet adhesion and vascular resistance. Tempol can scavenge the reactive oxygen species (ROS) that induce tissue injury. As xenograft rejection attenuates endogenous NO production and generates ROS, we evaluated the potential effect of an NO donor (SIN-1, 3-morpholinosydnonimine) and tempol on hyperacute xenograft dysfunction using an ex vivo porcine lung perfusion model., Methods: For the evaluation of von Willebrand factor (vWF) secretion, human endothelial cells were stimulated with thrombin. Porcine lungs were perfused with either fresh human whole blood (unmodified control group [n = 4]), SIN-1 (n = 4), or SIN and tempol (n = 4)., Results: SIN-1 and tempol significantly inhibited vWF secretion from endothelial cells in vitro. However, they did not suppress xenogeneic complement activation. In an ex vivo pulmonary perfusion model, SIN-1 improved pulmonary xenograft function by reducing pulmonary vascular resistance (PVR), inhibiting complement activation, and inhibiting thrombin generation. Combined treatment with tempol and SIN-1 potentiated PVR reduction, but slightly enhanced complement activation., Conclusions: An NO donor is expected to improve pulmonary xenograft function through inhibition of vWF secretion, vasoconstriction, thrombin generation, and indirectly through inhibition of complement activation. The additional effects of tempol on an NO donor were not considered significant in an ex vivo xenograft system., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by L-tyrosine, nitrite and (-)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H₂S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H₂S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H₂S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H₂S in inflammation., (Copyright © 2015 the American Physiological Society.)

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Excessive intracellular accumulation of zinc (Zn(2+)) is neurotoxic and contributes to a number of neuropathological conditions. Here, we investigated the protective effect of 3-morpholinosydnonimine (SIN-1) against Zn(2+)-induced neuronal cell death in differentiated PC12 cells. We found that Zn(2+)-induced PC12 cell death was reduced in a concentration-dependent manner by pretreatment with SIN-1. The intracellular accumulation of Zn(2+) was not affected by pretreatment with SIN-1, indicating that SIN-1-induced neuroprotection was not attributable to reduced influx of Zn(2+) into cells. SIN-1C, the stable decomposition product of SIN-1, failed to prevent Zn(2+)-induced cell death. Furthermore, the protective effect of SIN-1 against Zn(2+)-induced PC12 cell death was almost completely abolished by uric acid, a free radical scavenger, suggesting that reactive oxygen and nitrogen species generated by SIN-1 may contribute to the protective effect. SIN-1 prevented the inactivation of glutathione reductase (GR) and the increase in the ratio of oxidized glutathione/total glutathione (GSSG/total GSH) induced by Zn(2+). Addition of membrane permeable GSH ethyl ester (GSH-EE) to PC12 cells prior to Zn(2+) treatment significantly increased cell viability. We therefore conclude that SIN-1 may exert neuroprotective effect against Zn(2+)-induced cell death in differentiated PC12 cells by preventing inhibition of GR and increase in GSSG/total GSH ratio., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 μM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.

Chondrocyte senescence is associated with the aging and degeneration of cartilage, and eventually leads to joint destruction. The aim of this study was to elucidate the mechanisms responsible for the cytoprotective effects of heme oxygenase‑1 (HO‑1) on chondrocytes in cartilage. Chondrocyte senescence was induced using cilostazol and measured using a specific senescence‑associated β‑galactosidase (SA‑β‑gal) staining assay. Cilostazol altered the expression of type Ⅱ collagen and β‑catenin, which are phenotypic markers of the differentiation and dedifferentiation of chondrocytes. Cilostazol also significantly induced HO‑1 expression, and the induction of HO‑1 expression was affected by a significant increase in reactive oxygen species (ROS) production caused by cilostazol treatment. Of note, pre‑treatment with 3‑morpholinosydnonimine hydrochloride (SIN‑1), an inducer of HO‑1 expression, markedly attenuated cilostazol‑induced chondrocyte senescence, and thus, we examined whether HO‑1 directly modulates chondrocyte senescence induced by cilostazol. The upregulation of HO‑1 was found to suppress cilostazol‑induced cellular senescence. In addition, the inhibition of HO‑1 activity with the iron chelator, desferrioxamine (DFO), or HO‑1 siRNA increased cilostazol‑induced chondrocyte senescence. Based on these results, it can be concluded that HO‑1 is associated with the suppression of chondrocyte senescence, and that the enforced overexpression of HO‑1 protects chondrocytes against stress‑induced senescence.

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

Mesenchymal stem cells (MSCs) are a heterogeneous population of cells that proliferate in vitro as plastic-adherent cells, have a fibroblast-like morphology, form colonies in vitro and can differentiate into bone, cartilage and fat cells. The abundance, ease and repeatable access to subcutaneous adipose tissue and the simple isolation procedures provide clear advantages for the use of human adipose tissue-derived mesenchymal stem cells (hASDCs) in clinical applications. We screened microRNAs (miRNAs) that affected the proliferation and survival of hADSCs. Transfection of miR-302d mimic increased cell proliferation and protected cells from oxidant-induced cell death in hADSCs, which was supported by flow-cytometric analysis. miR-302d did not affect the expression of Bcl-2 family members or anti-oxidant molecules. The Nrf2-Keap1 system, which is one of the major mechanisms for the cellular defense against oxidative stress, was not altered by transfection of miR-302d mimic. To identify the target of the miR-302d actions on proliferation and survival of hADSCs, a microarray analysis was performed using miR-302d-overexpressing hADSCs. Real-time PCR analysis showed that transfection of miR-302d mimic inhibited the CDKN1A and CCL5 expression. Downregulation of CDKN1A with a specific siRNA mimicked the effect of miR-302d on hADSCs proliferation, but did not affect miR-302d-induced cell survival. Downregulation of CCL5 protected oxidant-induced cell death as miR-302d, inhibited oxidant-induced reactive oxygen species (ROS) generation and the addition of recombinant CCL5 inhibited the protective action of miR-302d on oxidant-induced cell death. This study indicates that miR-302 controls proliferation and cell survival of hADSCs through different targets and that this miRNA can be used to enhance the therapeutic efficacy of hADSCs transplantation in vivo.

Background and Purpose: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC)., Experimental Approach: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured., Key Results: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO., Conclusion and Implications: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases., (© 2014 The British Pharmacological Society.)

Background: Growing evidence indicates that patients with complex regional pain syndrome (CRPS) exhibit tissue abnormalities caused by microvascular dysfunction in the blood vessels of skin, muscle, and nerve. We tested whether topical combinations aimed at improving microvascular function would relieve allodynia in an animal model of CRPS. We hypothesized that topical administration of either α2-adrenergic (α2A) receptor agonists or nitric oxide (NO) donors given to increase arterial blood flow, combined with either phosphatidic acid (PA) or phosphodiesterase (PDE) inhibitors to increase capillary blood flow, would effectively reduce allodynia and signs of microvascular dysfunction in the animal model of chronic pain., Methods: Mechanical allodynia was induced in the hindpaws of rats with chronic postischemia pain (CPIP). Allodynia was assessed before and after topical application of vehicle, single drugs or combinations of an α2A receptor agonist (apraclonidine) or an NO donor (linsidomine), with PA or PDE inhibitors (lisofylline, pentoxifylline). A topical combination of apraclonidine + lisofylline was also evaluated for its effects on a measure of microvascular function (postocclusive reactive hyperemia) and tissue oxidative capacity (formazan production by tetrazolium reduction) in CPIP rats., Results: Each of the single topical drugs produced significant dose-dependent antiallodynic effects compared with vehicle in CPIP rats (N = 30), and the antiallodynic dose-response curves of either PA or PDE inhibitors were shifted 5- to 10-fold to the left when combined with nonanalgesic doses of α2A receptor agonists or NO donors (N = 28). The potent antiallodynic effects of ipsilateral treatment with combinations of α2A receptor agonists or NO donors with PA or PDE inhibitors were not reproduced by the same treatment of the contralateral hindpaw (N = 28). Topical combinations produced antiallodynic effects lasting up to 6 hours (N = 15) and were significantly enhanced by low-dose systemic pregabalin in early, but not late, CPIP rats (N = 18). An antiallodynic topical combination of apraclonidine + lisofylline was also found to effectively relieve depressed postocclusive reactive hyperemia in CPIP rats (N = 61) and to increase formazan production in postischemic tissues (skin and muscle) (N = 56)., Conclusions: The present results support the hypothesis that allodynia in an animal model of CRPS is effectively relieved by topical combinations of α2A receptor agonists or NO donors with PA or PDE inhibitors. This suggests that topical treatments aimed at improving microvascular function by increasing both arterial and capillary blood flow produce effective analgesia for CRPS.

The peroxynitrite free radical (ONOO(-)) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO(-), induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO(-) on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd(2+). Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO(-)-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca(2+) channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO(-) at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.

Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

Objectives: It is aimed to investigate the effects of guanylyl cyclase activation and inhibition on acute morphine antinociception and the development of tolerance to its effect., Background: Nitric oxide-soluble guanylyl cyclase signal transduction cascade suggested to play an important role in the development of tolerance to antinociceptive effects of morphine., Methods: Nociception was evaluated by tail flick and hot plate tests in male Wistar rats. The analgesic effects of intraperitoneal protoporphyrin IX (PPIX; an activator of soluble guanylyl cyclase), 3-morpholinosydnonimine hydrochloride (SIN-1; NO donor and activator of guanylyl cyclase), S-Nitroso-N-acetylpenicillamine (SNAP; an activator of guanylyl cyclase), 3,3-Bis (amino ethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18; NO donor activating guanylyl cyclase) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of guanylyl cyclase) alone or in combination with subcutaneous morphine injection were evaluated. Their effects on morphine tolerance development were evaluated by giving these agents 20 minutes prior to twice daily morphine injection during tolerance development for 5 days. On day 6, the expression of morphine tolerance was determined., Results: PPIX, SIN-1, SNAP and NOC-18 significantly increased expression of morphine tolerance while ODQ decreased., Conclusion: These data suggested that sGC activators have a significant role in tolerance to the analgesic effect of morphine (Tab. 1, Fig. 4, Ref. 29).

Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), a peroxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole-cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate independent, whereas in the ventricle APD, prolongation was rate dependent. In addition to prolongation of the action potential, beat-to-beat variability of repolarization was significantly increased in ventricular but not in atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1-induced increase in ventricular beat-to-beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.

Reactive oxygen species (ROS)-induced pancreatic β cell death affects insulin secretion and is important in the pathogenesis of diabetes. Lagerstroemia speciosa, a traditional folk medicine, has been used for t he prevention and treatment of diabetes. However, whether Lagerstroemia speciosa has a cytoprotective effect on pancreatic β cells remains to be elucidated. The present study aimed to investigate the cytoprotective effects of hot water extracts from Lagerstroemia speciosa leaves (LWE) on 3-morpholinosydnonimine (SIN-1)-induced oxidative damage in Syrian hamster pancreatic insulinoma HIT-T15 cells. The HIT-T15 cells were first treated with SIN-1 (50 µM) for 24 h and then co-incubated with LWE for 48 h. SIN-1 significantly decreased HIT-T15 cell viability (P<0.05); however, LWE did not exert a significant cytotoxic effect and increased the viability of HIT-T15 cells in a dose‑dependent manner. To further investigate the protective effects of LWE on SIN-1‑induced oxidative stress in HIT-T15 cells, the cellular levels of ROS, lipid peroxidation and endogenous antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px), were determined. LWE decreased the intracellular levels of ROS and lipid peroxidation, and increased the activities of antioxidant enzymes. These results suggest that LWE has a cytoprotective effect against SIN-1‑induced oxidative stress in HIT-T15 cells through the inhibition of lipid peroxidation, a decrease in ROS levels and an increase in antioxidant enzyme activity. In addition, LWE increased insulin secretion in SIN-1-treated HIT-T15 cells. Our results suggested that LWE were effective in the treatment of diabetes. Further studies are required to study the anti-diabetic molecular mechanism in a cell model.

Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) and it is a new area of research in higher plants. Previously, it was demonstrated that the exposition of sunflower (Helianthus annuus L.) seedlings to high temperature (HT) caused both oxidative and nitrosative stress. The nitroproteome analysis under this stress condition showed the induction of 13 tyrosine-nitrated proteins being the carbonic anhydrase (CA) one of these proteins. The analysis of CA activity under high temperature showed that this stress inhibited the CA activity by a 43%. To evaluate the effect of nitration on the CA activity in sunflower it was used 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent. Thus the CA activity was inhibited by 41%. In silico analysis of the pea CA protein sequence suggests that Tyr(205) is the most likely potential target for nitration., (Copyright © 2012 Elsevier Inc. All rights reserved.)