Reactive Peroxynitrite Research Articles

Peroxynitrite mediates active site tyrosine nitration in manganese superoxide dismutase. Evidence of a role for the carbonate radical anion.

Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative, and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (k(cat) = 9.3 × 10(2) M(-1) s(-1)) indicates that the mechanism involves redox cycling between Mn(2+) and Mn(3+), similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were the most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, in both the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3(-)) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3(-).

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif−/−) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.

Amyloid-β Peptide Fibrils Induce Nitro-Oxidative Stress in Neuronal Cells

Different mechanisms including oxidative stress are proposed for amyloid-β peptide (Aβ) neurotoxicity, and here we contribute to demonstrate that nitro-oxidative stress is playing a key role. Yeasts are a well-known model for H2O2 toxicity. Interestingly, yeast cell wall prevents interaction of Aβ fibrils with membrane receptors or calcium channels and we found a significant viability reduction in yeasts when challenged with Aβ fibrils. Furthermore, iron and copper chelators, as well as the antioxidants glutathione and trolox, were neuroprotective on neuroblastoma cells and mouse hippocampal neurons challenged with Aβ fibrils. Glutathione prevents the oxidation, glycation and nitrotyrosination of cell proteins induced by Aβ. Trolox protected neurons in cell viability studies, maintaining the vesicular transport integrity and preventing the trigger of apoptotic mechanisms. Interestingly, we have also found that brain derived neuronal factor (BDNF) and neurotrophin-3 (NT-3) were able to protect mouse hippocampal and cortical neurons against H2O2 and Aβ fibrils. Considering that superoxide anion, produced by Aβ cell damage, and nitric oxide, whose production is altered in AD, react to form the highly reactive peroxynitrite anion, we studied the role of trolox to ameliorate the peroxynitrite cell damage. Finally, one of the major proteins to be nitrotyrosinated in AD, the triose phosphate isomerase (TPI) was assayed searching for a denitrase activity that could reverse intracellular nitrotyrosination. We have found that human neuroblastoma SH-SY5Y cells express a constitutive denitrase activity that partially denitrated nitro-TPI. Altogether, our results support a key role of nitro-oxidative stress in the neuronal damage induced by Aβ fibrils.

Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function

The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.

NO synthase isoforms specifically modify peroxynitrite reactivity

Nitric oxide synthases (NOSs) are multi-domain hemothiolate proteins that are the sole source of nitric oxide (NO) in mammals. NOSs can also be a source or a sink for peroxynitrite (PN), an oxidant that is suspected to be involved in numerous physiopathological processes. In a previous study, we showed that the oxygenase domain of the inducible NOS (iNOSoxy) reacts with PN and changes its oxidative reactivity [Maréchal A, Mattioli TA, Stuehr DJ & Santolini J (2007) J Biol Chem 282, 14101-14112]. Here we report a similar analysis on two other NOS isoforms, neuronal NOS (nNOS) and a bacterial NOS-like protein (bsNOS). All NOSs accelerated PN decomposition, with accumulation of a similar heme intermediate. The kinetics of PN decomposition and heme transitions were comparable among NOSs. However, their effects on PN reactivity differ greatly. All isoforms suppressed PN two-electron oxidative activity, but iNOSoxy enhanced PN one-electron oxidation and nitration potencies, the oxygenase domain of nNOS (nNOSoxy) affected them minimally, and bsNOS abolished all PN reactivities. This led to the loss of both NOS and PN decomposition activities for nNOSoxy and iNOSoxy, which may be linked to the reported alterations in their electronic absorption spectra. Bacterial bsNOS was affected to a lesser extent by reaction with PN. We propose that these differences in PN reactivity among NOSs might arise from subtle differences in their heme pockets, and could reflect the physiological specificity of each NOS isoform, ranging from oxidative stress amplification (iNOS) to detoxification (bsNOS).

Restraint stress up-regulates lectin-like oxidized low-density lipoprotein receptor-1 in aorta of apolipoprotein E-deficient mice

Psychological stress is a risk factor for cardiovascular disease including atherosclerosis, but the mechanisms are unknown. The vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in vascular pathology and early atherogenesis. We hypothesized that LOX-1 is up-regulated by psychological stress via the formation of oxygen-derived free radicals, and that treatment with EUK-8 (a superoxide dismutase and catalase mimetic) prevents production of oxygen-derived free radicals and leads to reduced expression of LOX-1 in the vascular wall. As a model for psychological stress, we exposed male apolipoprotein E-deficient mice to repeated restraint stress by placement in a conical tube for 2 h per day for 14 consecutive days. Stressed and control mice were treated with EUK-8 (n = 4–5) or vehicle (n = 4–5). Reactive oxygen species and peroxynitrite levels, as detected by oxidative fluorescence microscopy, were increased in the aortic root of mice exposed to stress compared to those of controls by 212 ± 22% (mean ± SEM; p < 0.001) and 110 ± 6% (p < 0.001), respectively. LOX-1, as detected by immunohistochemistry, was increased by 443 ± 63% in stressed mice compared to control mice (p < 0.001). EUK-8 reduced reactive oxygen species, peroxynitrite, and LOX-1 levels in stressed mice compared to vehicle-treated stressed mice. To conclude, LOX-1 induced by reactive oxygen species and/or peroxynitrite could be one mechanism by which stress promotes cardiovascular disease.

A miR-thless Perspective

Asymmetrical dimethylarginine (ADMA) is an endogenous derivative of the amino acid l-arginine that is formed by the proteolytic degradation of methylated protein residues. ADMA plasma levels are increased in patients with cardiovascular risk factors, although the mechanism underlying the increase may vary among individual risk factors: several enzymes involved in l-arginine methylation, demethylation, and ADMA transport are differentially regulated. It is generally believed that ADMA acts as a biologically significant endogenous inhibitor of the endothelial nitric oxide synthase (eNOS). The plasma concentrations of ADMA are, however, approximately 2 orders of magnitude lower than those of the eNOS substrate l-arginine. Thus, it is unlikely that a robust competitive antagonism to l-arginine at eNOS with a pronounced reduction in eNOS activity is the primary mode of action of ADMA.1 Rather, ADMA will induce a slight reduction of NO formation that appears to be sufficient to initiate processes that ultimately result in a massive breakdown of NO production. For example, ADMA promotes eNOS uncoupling, which, through a vicious cycle of reactive oxygen species (ROS) formation, tetrahydrobiopterin oxidation, and peroxynitrite formation, leads to overt endothelial dysfunction and an impaired vascular regenerative capacity.2 Moreover, it has been observed that ADMA has prooxidative and proinflammatory effects in cells lacking NO formation. This NO-independent component of the action of ADMA is mediated, at least in part, by the activation of the transcription factor nuclear factor κB.3–5 Bone marrow–derived progenitor cells, referred to as angiogenic progenitor cells (APCs), have gained considerable attention as a proangiogenic element. Despite ongoing discussions about the true nature of APCs, their …

5-Hydroxytrytophan Inhibitstert-Butylhydroperoxide (t-BHP)-Induced Oxidative Damage via the Suppression of Reactive Species (RS) and Nuclear Factor-κB (NF-κB) Activation on Human Fibroblast

5-Hydroxytryptophan (5HTP), an analogue of tryptophan, is a precursor of serotonin that also has effective antioxidative and anti-apoptotic properties (1) . However, the cellular mechanisms underlying these properties of 5HTP have not been explored. In this study, we tested the hypothesis that 5HTP exerts its antioxidative action against oxidative stress and inflammation by suppressing the activation of the key pro-inflammatory transcriptional pathways, p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kappaB (NF-kappaB). The study was carried out using human fibroblast cells that were challenged by tert-butylhydroperoxide (t-BHP)-induced oxidative damage. Results show that 5HTP significantly reduced t-BHP-induced oxidative damage in human fibroblast cells, as determined by cell cytotoxicity, intracellular reactive species (RS) and peroxynitrite (ONOO(-)) generation, and inducible nitric oxide synthase expression. Moreover, 5HTP protected human fibroblast cells against t-BHP-induced oxidative DNA damage, as determined by 4,6-diamidino-2-phenlylindole (DAPI) staining. Pretreatment of human fibroblast cells with 5HTP also dose-dependently inhibited glutathione (GSH) depletion, indicating that it protects cells against t-BHP-induced oxidative damage. Western blot analysis revealed that 5HTP also markedly increased Bcl-2 expression and suppressed both p38MAPK and NF-kappaB activation in the t-BHP-treated human fibroblast cells. When these results are taken together, they strongly indicate that 5HTP has beneficial and protective effects against t-BHP-induced cell death in vitro, as demonstrated by its antioxidative and anti-inflammatory actions. Data further showed that the protective mechanisms underlying the actions of 5HTP against oxidative stress-induced damage are associated with RS/ONOO(-) scavenging and the inhibition of lipid peroxidation and GSH depletion.

Mitochondrial function and dysfunction in sepsis

Mitochondria are the key source of cellular ATP and their structure and function are markedly affected by pathophysiologic processes associated with the host's response to invading pathogens. In particular, the highly reactive compound peroxynitrite, generated by the reaction of nitric oxide and superoxide anions, inhibits mitochondrial enzymes and damages lipids, proteins, and nucleic acids. Enhanced oxidative stress induces DNA strand breaks that are repaired by activation of poly(ADP-ribose)polymerase (PARP). This process consumes large amounts of nicotinamide adenine dinucleotide (NAD(+)) leading to cellular NAD(+) depletion that impairs flux of reducing equivalents into the respiratory chain and also further promotes inflammation. In experimental studies, novel therapeutic strategies that aim to ameliorate the host's pathogen response or to modulate intracellular signaling events related to oxidative stress protected mitochondrial function and preserved cellular respiration ultimately leading to improved organ function.

Hydroquinone modulates reactivity of peroxynitrite and nitric oxide production

Peroxynitrite (ONOO(-)), a potent cytotoxic oxidant formed by the reaction of nitric oxide ((.-)NO) and superoxide radical ((.-)O(2)(-)), may be rapidly lethal in a cellular milieu due to oxidization and nitration processes. In the present study, hydroquinone displayed strong ONOO(-) scavenging activity and inhibitory effect on NO production in murine macrophage RAW264.7 cells. Hydroquinone strongly scavenged ONOO(-)induced dihydrorhodamine 123 oxidation in a dose-dependent manner compared with other reactive species such as (.-)O(2)(-) and (.-)NO. Hydroquinone also decreased levels of ONOO(-) induced nitrotyrosine of glutathione reductase and consequently prevented the enzyme from ONOO(-) induced damage. Furthermore, hydroquinone suppressed NO production, a cellular pathway for ONOO(-) formation, in lipopolysaccharide-activated RAW264.7 cells via inhibition of inducible NO synthase expression. The inhibitory effect by hydroquinone seems to be mediated by interruption of lipopolysaccharide-induced signalling such as activation of nuclear factor-kappaB and extracellular signalrelated kinases 1 and 2. The results suggest that hydroquinone may potently modulate reactivity of ONOO(-) and may therefore be a useful agent against ONOO(-) mediated diseases.

Attenuation of oxidative stress and inflammation by gravinol in high glucose-exposed renal tubular epithelial cells

Gravinol, a proanthocyanidin from grape seeds, has polyphenolic properties with powerful anti-oxidative effects. Although, increasing evidence strongly suggests that polyphenolic antioxidants suppress diabetic nephropathy that is causally associated with oxidative stress and inflammation, gravinol's protective action against diabetic nephropathy has not been fully explored to date. In the current study, we investigated the protective action of gravinol against oxidative stress and inflammation using the experimental diabetic nephropathy cell model, high glucose-exposed renal tubular epithelial cells. To elucidate the underlying actions of gravinol, several oxidative and inflammatory markers were estimated. Included are measurements of lipid peroxidation, total reactive species (RS), superoxide ( O 2), nitric oxide (NO ), and peroxynitrite (ONOO −), as well as nuclear factor-kappa B (NF-κB) nuclear translocation. Results indicate that gravinol had a potent inhibitory action against lipid peroxidation, total RS, O 2, NO , ONOO −, the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and more importantly, against NF-κB nuclear translocation. We propose that gravinol's strong protective effect against high glucose-induced renal tubular epithelial cell damage attenuates diabetic nephropathy by suppressing oxidative stress and inflammation.

Mechanism of light-induced oxidation of nitrosylmyoglobin

Exposure of nitrosylmyoglobin (MbFe IINO), to light in the presence of oxygen increases rate of oxidation still with metmyoglobin (MbFe III) as main product, as shown by electron paramagnetic resonance and visible absorption spectroscopy. Despite previous suggestions of interfering radical species, capable of initiating lipid oxidation in cured meat, no radical adducts were detected during photolysis in spin trapping experiments. Flash photolysis showed separation of myoglobin (MbFe II) and nitric oxide (NO ) after excitation, followed by fast rebinding under anaerobic conditions. In the presence of oxygen, nitric oxide rebinding was inhibited suggesting formation of a nitrosyldioxyl-radical (ONOO ). As NO failed to diffuse into bulk solution, ONOO formation is proposed to occur in the heme cavity in competition with NO rebinding and, to a lesser degree, O 2 binding to yield MbFe IIO 2. Subsequent oxidation of MbFe II to MbFe III by ONOO was expected to result in the highly reactive peroxynitrite intermediate (ONOO −), of which occurrence would be characterized by typical chemical footprints, such as nitrated tyrosine or tryptophan residues. However, results from direct infusion mass spectrometry suggested neat conversion from nitrosylmyoglobin to metmyoglobin and questioned the existence of ONOO − as reaction product. Catalytic activity of the resulting ferric centre in MbFe III, however, induces fast isomerisation of ONOO − to the stable nitrate, which was identified directly as ion m/ z 62 or indirectly by adduct formation with MbFe III.

Signaling of reactive oxygen and nitrogen species in Diabetes mellitus.

Disorder of physiological signaling functions of reactive oxygen species(ROS) superoxide and hydrogen peroxide and reactive nitrogen species (RNS) nitric oxide and peroxynitrite is an important feature of diabetes mellitus type 1 and type 2. It is now known that hyperglycemic conditions of cells are associated with the enhanced levels of ROS mainly generated by mitochondria and NADPH oxidase. It has been established that ROS stimulate many enzymatic cascades under normal physiological conditions, but hyperglycemia causes ROS overproduction and the deregulation of ROS signaling pathways initiating the development of diabetes mellitus. On the other hand the deregulation of RNS signaling leads basically to a decrease in NO formation with subsequent damaging disorders. In the present work we will consider the pathological changes of ROS and RNS signaling in enzyme/gene regulated processes catalyzed by protein kinases C and B (Akt/B), phosphatidylinositol 3′-kinase (PI3-kinase), extracellular signal-regulated kinase 1/2 (ERK1/2) and some others. Furthermore we will discuss a particularly important role of several ROS-regulated genes and adapter proteins such as the p66shc, FOXO3a and Sirt2. The effects of low and high ROS levels in diabetes will be also considered. Thus the regulation of damaging ROS levels in diabetes by antioxidants and free radical scavengers must be one of promising treatment of this disease, however, because of the inability of traditionalantioxidative vitamin E and C to interact with superoxide and hydrogen peroxide,new free radical scavengers such as flavonoids, quinones and synthetic mimetics of superoxide dismutase (SOD) should be intensively studied.

The Anti-apoptotic Action of 5-Hydroxyindole: Protection of Mitochondrial Integrity

5-Hydroxyindole (5HI), a metabolite of tryptophan, is involved in learning and memory, central neuron system regulation, and anti-oxidant activity. However, its protective action in mitochondrial function is not clear. Here, we tested whether 5HI protects against tert-butylhydroperoxide (t-BHP)-induced oxidative damage and mitochondrial dysfunction in human fibroblast cells. 5HI significantly suppressed t-BHP-induced cytotoxicity as determined by intracellular reactive species generation, lipid peroxidation, glutathione depletion, and peroxynitrite (ONOO(-)) generation. In addition, 5HI reduced t-BHP-induced DNA condensation. Pretreatment with 5HI significantly restored mitochondrial membrane potential (Deltapsim), suggesting that it protected cells against t-BHP-induced apoptosis. Western blot analysis also revealed that 5HI markedly inhibited cytochrome c release and caspase-3 activation, but not caspase-9 activation. Our data suggest that 5HI protects cells by attenuating oxidative stress and consequently protects against mitochondrial dysfunction.

Effect of curcumin on the nitric oxide induced structural and functional modifications of high molecular mass goat brain cystatin

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Under physiological conditions nitric oxide can be found in almost all the tissues, but under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Our present study was designed to investigate the protective effect of curcumin against NO induced damage of HM-GBC. NO caused intensive structural and functional damage of HM-GBC, resulting in 89% loss of its antiproteolytic activity after 2 h of incubation. Structural damage occurs in the form of protein degradation. Curcumin significantly protected HM-GBC against this damage. This suggests that curcumin has a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs.

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.

Protection against Oxidative Stress, Inflammation, and Apoptosis of High-Glucose-Exposed Proximal Tubular Epithelial Cells by Astaxanthin

Astaxanthin is a carotenoid with powerful antioxidant properties that exists naturally in various plants, algae, and seafood. The purpose of the present study is to examine the protective action of astaxanthin against high-glucose-induced oxidative stress, inflammation, and apoptosis in proximal tubular epithelial cells (PTECs). To assess the efficacy of astaxanthin, several key markers and activities were measured, including lipid peroxidation, total reactive species (RS), superoxide (*O(2)), nitric oxide (NO*), and peroxynitrite (ONOO(-)), as well as expressions of inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-kappaB) nuclear translocation, and levels of Bcl2/Bax protein. Results showed that astaxanthin effectively suppressed lipid peroxidation, total RS, *O(2), NO*, ONOO(-), iNOS and COX-2 protein levels, NF-kappaB nuclear translocation, and pro-apototic Bax, whereas it increased anti-apoptotic Bcl2 protein levels. On the basis of these findings, it was concluded that in PTECs, astaxanthin has a protective efficacy against several deleterious effects caused by high glucose exposure and proposed that astaxanthin should be explored further as a potential antidiabetic remedy for the treatment of diabetic nephropathy.

Comprehensive evaluation of antioxidant activity for various substances with 5-axe cobweb chart

Antioxidant activities of various substances against peroxynitrite and peroxyl radical generated from 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) were evaluated by the previously proposed method [Terashima, M., Nakatani, I., Harima, A., Nakamura, S., & Shiiba M. (2007). New method to evaluate water-soluble antioxidant activity based on protein structure change. Journal of Agricultural and Food Chemistry, 55(1), 165–169] using myoglobin as a probe. Activities of several antioxidants against five different reactive oxygen species, DPPH, hypochlorite ion, hydroxyl radical, peroxynitrite, and peroxyl radical, were characterised comprehensively by 5-axe cobweb charts. For example, Trolox, ascorbic acid, and ferulic acid show similar activities against DPPH radicals (7–10 mM VC), the shapes of the 5-axe cobweb charts are different from each others. This clear-cut expression would be useful to predict probable physiological effects of various antioxidants and to compare quality of antioxidants contained in food materials and food products.

NF-kappaB-induced oxidative stress contributes to mitochondrial and cardiac dysfunction in type II diabetes.

Inflammatory molecules and their transcription factor, nuclear factor kappa-B (NF-kappaB), are thought to play important roles in diabetes-induced cardiac dysfunction. Here, we investigated the effects of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, in diabetic mice. Obese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-kappaB activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-kappaB, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups. This study demonstrates that NF-kappaB blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.

Effect of curcumin on the nitric oxide induced structural and functional modifications of high molecular mass goat brain cystatin

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Under physiological conditions nitric oxide can be found in almost all the tissues, but under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Our present study was designed to investigate the protective effect of curcumin against NO induced damage of HM-GBC. NO caused intensive structural and functional damage of HM-GBC, resulting in 89% loss of its antiproteolytic activity after 2 h of incubation. Structural damage occurs in the form of protein degradation. Curcumin significantly protected HM-GBC against this damage. This suggests that curcumin has a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs.