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Sample records for nadph oxidase activity

  1. BRAINA JOURNAL OF NEUROLOGY NADPH oxidase expression in active multiple

    E-print Network

    Hayar, Abdallah

    BRAINA JOURNAL OF NEUROLOGY NADPH oxidase expression in active multiple sclerosis lesions.lassmann@meduniwien.ac.at Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions

  2. Paradoxical Activation of Endothelial Nitric Oxide Synthase by NADPH oxidase

    PubMed Central

    Zhang, Qian; Malik, Pulkit; Pandey, Deepesh; Gupta, Sonali; Jagnandan, Davin; de Chantemele, Eric Belin; Banfi, Botond; Marrero, Mario B.; Rudic, R Daniel; Stepp, David W.; Fulton, David J.R.

    2009-01-01

    Objectives Increased formation of reactive oxygen species (ROS) has been identified as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling endothelial nitric oxide synthase (eNOS). However, the specific contribution of ROS to endothelial function is not well understood. Methods and Results A major source of intracellular ROS is the NADPH oxidase (Nox) family of enzymes. The goal of the current study was to directly assess the contribution of NADPH oxidase derived superoxide to eNOS function by expressing Nox5, a single gene product that constitutively produces superoxide within cells. Paradoxically, we found that instead of inhibiting eNOS, co-expression of Nox5 increased NO release from both bovine and human endothelial cells. To establish the functional significance of this observation in intact blood vessels, the endothelium of mouse aorta was transduced with Nox5 or control adenoviruses. Nox5 potently inhibited Ach-induced relaxation and potentiated contractile responses to phenylephrine. In precontracted aortae, acute exposure to superoxide dismutase induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. Conclusions These findings suggest that ROS inhibit eNOS function via consumption of NO rather than direct inhibition of enzymatic activity. PMID:18556569

  3. Enhancing plasma membrane NADPH oxidase activity increases current output by diatoms in biophotovoltaic devices

    E-print Network

    Laohavisit, Anuphon; Anderson, Alexander; Bombelli, Paolo; Jacobs, Matthew; Howe, Christopher J.; Davies, Julia M.; Smith, Alison G.

    2015-08-31

    Biophotovoltaic (BPV) devices employ the photosynthetic activity of microalgae or cyanobacteria to harvest light energy and generate electrical current directly as a result of the release of electrons from the algal cells. NADPH oxidases (NOX...

  4. Complement-dependent NADPH oxidase enzyme activation in renal ischemia/reperfusion injury.

    PubMed

    Simone, S; Rascio, F; Castellano, G; Divella, C; Chieti, A; Ditonno, P; Battaglia, M; Crovace, A; Staffieri, F; Oortwijn, B; Stallone, G; Gesualdo, L; Pertosa, G; Grandaliano, G

    2014-09-01

    NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker ?-smooth muscle actin (?-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as ?-SMA expression and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and ?-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting. PMID:25017967

  5. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  6. Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.

    PubMed Central

    Dekker, L V; Leitges, M; Altschuler, G; Mistry, N; McDermott, A; Roes, J; Segal, A W

    2000-01-01

    We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils. PMID:10727429

  7. Inhibition of NAD(P)H Oxidase Activity Blocks Vascular Endothelial Growth Factor Overexpression and Neovascularization during Ischemic Retinopathy

    PubMed Central

    Al-Shabrawey, Mohamed; Bartoli, Manuela; El-Remessy, Azza B.; Platt, Daniel H.; Matragoon, Sue; Behzadian, M. Ali; Caldwell, Robert W.; Caldwell, Ruth B.

    2005-01-01

    Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy. PMID:16049343

  8. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-?B- (NF-?B-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) ? in BV-2 cells. We also observed that NF-?B activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-?B activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-?B to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  9. Low Extracellular Zinc Increases Neuronal Oxidant Production Through NADPH Oxidase and Nitric Oxide Synthase Activation

    PubMed Central

    Aimo, Lucila; Cherr, Gary N.; Oteiza, Patricia I.

    2012-01-01

    A decrease in zinc (Zn) levels increases the production of cell oxidants, affects the oxidant defense system and triggers oxidant sensitive signals in neuronal cells. However, the underlying mechanisms are still unclear. This work tested the hypothesis that the increase in neuronal oxidants that occurs when cellular Zn decreases is mediated by the activation of the NMDA receptor. Differentiated PC12 cells were cultured in control, Zn-deficient or Zn-repleted media. The incubation in Zn deficient media led to a rapid increase in cellular calcium levels, which was prevented by a NMDA receptor antagonist (MK-801). Cellular calcium accumulation was associated with NADPH oxidase and nitric oxide synthase (NOS) activation, an increase in cell oxidant levels, and an associated activation of a redox-sensitive signal (AP-1). In cells incubated in the Zn deficient medium, NADPH oxidase activation was prevented by MK-801 and by a protein kinase C inhibitor. The rise in cell oxidants was prevented by inhibitors of NADPH oxidase, of the NOS and by MK-801. A similar pattern of inhibitor action was observed for zinc deficiency-induced AP-1 activation. Results demonstrate that a decrease in extracellular Zn leads to an increase in neuronal oxidants through the activation of the NMDAR that leads to calcium influx and to a calcium-mediated activation of protein kinase C/NADPH oxidase and NOS. Changes in extracellular Zn concentrations can be sensed by neurons, which using reactive oxygen and nitrogen species as second messengers, can regulate signaling involved in neuronal development and function. PMID:20211250

  10. Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

    PubMed Central

    Gan, Xiaoliang; Xing, Dandan; Su, Guangjie; Li, Shun; Luo, Chenfang; Irwin, Michael G.; Xia, Zhengyuan; Li, Haobo; Hei, Ziqing

    2015-01-01

    Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and ?-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation. PMID:26246867

  11. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    PubMed

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1?M fMLF and 1?M WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. PMID:24880063

  12. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    SciTech Connect

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-04-15

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

  13. NADPH oxidase activity and reactive oxygen species production in brain and kidney of adult male hypertensive Ren-2 transgenic rats.

    PubMed

    Vokurková, M; Rauchová, H; ?ezá?ová, L; Van??ková, I; Zicha, J

    2015-12-29

    Hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play an important role in brain control of blood pressure (BP). One of the important mechanisms involved in the pathogenesis of hypertension is the elevation of reactive oxygen species (ROS) production by nicotine adenine dinucleotide phosphate (NADPH) oxidase. The aim of our present study was to investigate NADPH oxidase-mediated superoxide (O(2)(-)) production and to search for the signs of lipid peroxidation in hypothalamus and medulla oblongata as well as in renal medulla and cortex of hypertensive male rats transgenic for the murine Ren-2 renin gene (Ren-2 TGR) and their age-matched normotensive controls - Hannover Sprague Dawley rats (HanSD). We found no difference in the activity of NADPH oxidase measured as a lucigenin-mediated O(2)(-) production in the hypothalamus and medulla oblongata. However, we observed significantly elevated NADPH oxidase in both renal cortex and medulla of Ren-2 TGR compared with HanSD. Losartan (LOS) treatment (10 mg/kg body weight/day) for 2 months (Ren-2 TGR+LOS) did not change NADPH oxidase-dependent O(2)(-) production in the kidney. We detected significantly elevated indirect markers of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) in Ren-2 TGR, while they were significantly decreased in Ren-2 TGR+LOS. In conclusion, the present study shows increased NADPH oxidase activities in renal cortex and medulla with significantly increased TBARS in renal cortex. No significant changes of NADPH oxidase and markers of lipid peroxidation were detected in the studied brain regions. PMID:26713567

  14. Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

    SciTech Connect

    Ahluwalia, Jatinder

    2008-01-11

    Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

  15. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  16. Activation of NADPH Oxidase 1 Increases Intracellular Calcium and Migration of Smooth Muscle Cells

    PubMed Central

    Zimmerman, Matthew C.; Takapoo, Maysam; Jagadeesha, Dammanahalli K.; Stanic, Bojana; Banfi, Botond; Bhalla, Ramesh C.; Miller, Francis J.

    2011-01-01

    Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium levels ([Ca2+]i). Using cultured SMCs from wild type (WT) and Nox1 null (Nox1?/y) mice, we confirmed that thrombin-dependent generation of ROS requires Nox1. Thrombin rapidly increased [Ca2+]i, as measured by fura-2 fluorescence ratio imaging, in WT but not Nox1 null SMCs. The increase in [Ca2+]i in WT SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca2+]i showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in WT and Nox1 null SMCs. To examine the effects of Nox1 on Ca2+ entry, cells were either bathed in Ca2+-free media or exposed to dihydropyridines to block L-type Ca2+ channel activity. Treatment with nifedipine or removal of extracellular Ca2+ reduced the thrombin-mediated increase of [Ca2+]i in WT SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca2+]i in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca2+ mobilization in SMCs, in part through regulation of Ca2+ influx, to thereby promote cell migration. PMID:21810651

  17. Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

    PubMed Central

    González-Perilli, Lucía; Álvarez, María Noel; Prolo, Carolina; Radi, Rafael; Rubbo, Homero; Trostchansky, Andrés

    2013-01-01

    Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species (ROS) under pro-inflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2·?), due to the NADPH dependent univalent reduction of oxygen to O2·? by the phagocytic NADPH-oxidase isoform (NOX2), being the activity of NOX2 the main source of O2·? in monocytes/macrophages. Since NOX2 and AA pathways are connected, we propose that AANO2can modulate macrophage activation by inhibiting O2·? formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex Red fluorescence and flow cytometry; this process also occurs in physiological mimic conditions within the phagosomes. AANO2 decreased O2·? production in a dose-(IC50= 4.1 ± 1.8 ?M AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g. p40phox and p47phox), as analyzed by immunoprecipitation and western blot. However, a reduction of the migration to the membrane of p47phox was obtained suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, where subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate treated mice. PMID:23318789

  18. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  19. Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

    PubMed

    Kassan, Modar; Ait-Aissa, Karima; Ali, Maha; Trebak, Mohamed; Matrougui, Khalid

    2015-10-01

    We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2. PMID:26036345

  20. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress. PMID:21651898

  1. Current status of NADPH oxidase research in cardiovascular pharmacology

    PubMed Central

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

    2013-01-01

    The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new inhibitors. All this knowledge and the research presently underway will likely result in development of new drugs for inhibition of NADPH oxidase and application of therapeutic approaches based on their action, for the treatment of cardiovascular disease in the next few years. PMID:23983473

  2. Seizure activity results in calcium- and mitochondria-independent ROS production via NADPH and xanthine oxidase activation

    PubMed Central

    Kovac, S; Domijan, A-M; Walker, M C; Abramov, A Y

    2014-01-01

    Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na+] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets. PMID:25275601

  3. Prolonged exposure to LPS increases iron, heme, and p22phox levels and NADPH oxidase activity in human aortic endothelial cells: Inhibition by desferrioxamine

    PubMed Central

    Li, Lixin; Frei, Balz

    2009-01-01

    Objective Vascular oxidative stress and inflammation are contributing factors in atherosclerosis. We recently found that the iron chelator, desferrioxamine (DFO), suppresses NADPH oxidase-mediated oxidative stress and expression of cellular adhesion molecules in mice treated with lipopolysaccharide (LPS). The objective of the present study was to investigate whether and how LPS and iron enhance, and DFO inhibits, NADPH oxidase activity in human aortic endothelial cells (HAEC). Methods and Results Incubation of HAEC for 24 hrs with 5 ?g/mL LPS led to a four-fold increase in NADPH oxidase activity, which was strongly suppressed by pretreatment of the cells for 24 hrs with 100 ?mol/L DFO. Incubating HAEC with LPS also significantly increased cellular iron and heme levels and mRNA and protein levels of p22phox, a heme-containing, catalytic subunit of NADPH oxidase. All of these effects of LPS on HAEC were strongly inhibited by DFO. Exposing HAEC to 100 mol/L iron (ferric citrate) for 48 hrs exerted similar effects as LPS, and these effects were strongly inhibited by co-incubation with DFO. Furthermore, neither LPS nor DFO affected mRNA and protein levels of p47phox, a non-heme containing, regulatory subunit of NADPH oxidase, or the mRNA level of NOX4, an isoform of the principal catalytic subunit of NADPH oxidase in endothelial cells. In contrast, heme oxygenase-1 was strongly suppressed by DFO, both in the absence and presence of LPS or iron. Conclusions Our data indicate that prolonged exposure to LPS or iron increases endothelial NADPH oxidase activity by increasing p22phox gene transcription and cellular levels of iron, heme, and p22phox protein. Iron chelation by DFO effectively suppresses endothelial NADPH oxidase activity, which may be helpful as an adjunct in reducing vascular oxidative stress and inflammation in atherosclerosis. PMID:19251588

  4. NADPH oxidase deficiency in X-linked chronic granulomatous disease.

    PubMed Central

    Hohn, D C; Lehrer, R I

    1975-01-01

    We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity. Images PMID:235560

  5. D1-Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells

    PubMed Central

    Li, Hewang; Han, Weixing; Villar, Van Anthony M.; Keever, Lindsay B.; Lu, Quansheng; Hopfer, Ulrich; Quinn, Mark T.; Felder, Robin A.; Jose, Pedro A.; Yu, Peiying

    2010-01-01

    NADPH oxidase (Nox)–dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D1-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58±3.3%) than in hypertensive rats (31±5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67phox in cells from normotensive rats. D1-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl–?-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats. PMID:19380616

  6. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    SciTech Connect

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin; Park, Ji-hoon; Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook; Kim, Soon Ha

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 ?M. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ? NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ? NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ? NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ? NecroX-7 effectively inhibited NADPH oxidase activation. ? Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

  7. The antioxidant activity of soursop decreases the expression of a member of the NADPH oxidase family.

    PubMed

    Zamudio-Cuevas, Y; Díaz-Sobac, R; Vázquez-Luna, A; Landa-Solís, C; Cruz-Ramos, M; Santamaría-Olmedo, M; Martínez-Flores, K; Fuentes-Gómez, A J; López-Reyes, A

    2014-02-01

    Cellular oxidative stress produced by an increase in free radicals is one of the factors that promote the development of chronic degenerative diseases; therefore, consuming natural antioxidants helps minimize their negative effects. This study evaluated the cytotoxicity of the soursop extract (Annona muricata), its cytoprotective capacity against oxidative stress induced by hydrogen peroxide, the inhibitory potential of reactive oxygen species (ROS), the molecular mechanism of its antioxidant action, and its capacity to repair cellular damage in the fibroblast cell line. The soursop extract proved not to be cytotoxic in fibroblast cultures and showed cytoprotective capacity against hydrogen peroxide-induced stress; in cell culture it reduced the generation of ROS significantly by inhibiting a sub-unit of the NADPH oxidase enzyme (p47phox). The soursop extract can prevent damage caused by cellular oxidants. PMID:24337133

  8. Role of NADPH Oxidase and Xanthine Oxidase in Mediating Inducible VT/VF and Triggered Activity in a Canine Model of Myocardial Ischemia

    PubMed Central

    Martins, James B.; Chaudhary, Ashok K.; Jiang, Shuxia; Kwofie, Michael; Mackie, Prescott; Miller, Francis J.

    2014-01-01

    Background: Ventricular tachycardia or fibrillation (VT/VF) of focal origin due to triggered activity (TA) from delayed afterdepolarizations (DADs) is reproducibly inducible after anterior coronary artery occlusion. Both VT/VF and TA can be blocked by reducing reactive oxygen species (ROS). We tested the hypothesis that inhibition of NADPH oxidase and xanthine oxidase would block VT/VF. Methods: 69 dogs received apocynin (APO), 4 mg/kg intraveneously (IV), oxypurinol (OXY), 4 mg/kg IV, or both APO and OXY (BOTH) agents, or saline 3 h after coronary occlusion. Endocardium from ischemic sites (3-D mapping) was sampled for Rac1 (GTP-binding protein in membrane NADPH oxidase) activation or standard microelectrode techniques. Results (mean ± SE, * p < 0.05): VT/VF originating from ischemic zones was blocked by APO in 6/10 *, OXY in 4/9 *, BOTH in 5/8 * or saline in 1/27; 11/16 VT/VFs blocked were focal. In isolated myocardium, TA was blocked by APO (10?6 M) or OXY (10?8 M). Rac1 levels in ischemic endocardium were decreased by APO or OXY. Conclusion: APO and OXY suppressed focal VT/VF due to DADs, but the combination of the drugs was not more effective than either alone. Both drugs inhibited ischemic Rac1 with inhibition by OXY suggesting ROS-induced ROS. The inability to totally prevent VT/VF suggests that other mechanisms also contribute to ischemic VT. PMID:25375191

  9. Attenuation of urokinase activity during experimental ischaemia protects the cerebral barrier from damage through regulation of matrix metalloproteinase-2 and NAD(P)H oxidase.

    PubMed

    Rakkar, Kamini; Srivastava, Kirtiman; Bayraktutan, Ulvi

    2014-06-01

    Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress. PMID:24649947

  10. New Insights into the Regulation of Neutrophil NADPH Oxidase Activity in the Phagosome: A Focus on the Role of Lipid and Ca2+ Signaling

    PubMed Central

    Bréchard, Sabrina; Plançon, Sébastien

    2013-01-01

    Abstract Significance: Reactive oxygen species, produced by the phagosomal NADPH oxidase of neutrophils, play a significant physiological role during normal defense. Their role is not only to kill invading pathogens, but also to act as modulators of global physiological functions of phagosomes. Given the importance of NADPH oxidase in the immune system, its activity has to be decisively controlled by distinctive mechanisms to ensure appropriate regulation at the phagosome. Recent Advances: Here, we describe the signal transduction pathways that regulate phagosomal NADPH oxidase in neutrophils, with an emphasis on the role of lipid metabolism and intracellular Ca2+ mobilization. Critical Issues: The potential involvement of Ca2+-binding S100A8 and S100A9 proteins, known to interact with the plasma membrane NADPH oxidase, is also considered. Future Directions: Recent technical progress in advanced live imaging microscopy will permit to focus more accurately on phagosomal rather than plasma membrane NADPH oxidase regulation during neutrophil phagocytosis. Antioxid. Redox Signal. 18, 661–676. PMID:22867131

  11. Female mice lacking active nadph-oxidase enzymes are protected against “western diet”--induced obesity and metabolic syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADPH oxidase (Nox) enzymes have been implicated in regulation of adipocyte differentiation and inflammation in a variety of tissues. We examined the effects of feeding AIN-93G or a “Western diet” (WD) (45% fat, 0.5% cholesterol) on development of obesity and “metabolic syndrome” in wild type (WT) m...

  12. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-?-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    SciTech Connect

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-10-15

    TNF-? plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-? induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-?B (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-? markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-?-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-?B (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-?-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-?-stimulated MAPKs and NF-?B activation. Thus, in H9c2 cells, we are the first to show that TNF-?-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-?B cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-?-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-? on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-? induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-? induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-? induces MMP-9 expression via a NADPH oxidase/ROS-dependent NF-?B signaling. • TNF-? activates MAPK phosphorylation through NADPH oxidase/ROS generation.

  13. Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

    SciTech Connect

    Pan Xinjuan; Dai Yujie; Li Xing; Niu Nannan; Li Wenjie; Liu Fangli; Zhao Yang; Yu Zengli

    2011-08-01

    Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.

  14. Inhibition of arsenic-induced rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-?/Smad activation.

    PubMed

    Pan, Xinjuan; Dai, Yujie; Li, Xing; Niu, Nannan; Li, Wenjie; Liu, Fangli; Zhao, Yang; Yu, Zengli

    2011-08-01

    Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30ppm) with or without GSE (100mg/kg, every other day by oral gavage) for 12months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-?1, type I procollagen (Coll-I) and ?-smooth muscle actin (?-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-?1-induced transactivation of the TGF-?-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-?1-induced mRNA expression of Coll-I and ?-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-?/Smad activation. PMID:21605584

  15. P2X7 receptor is critical in ?-synuclein--mediated microglial NADPH oxidase activation.

    PubMed

    Jiang, Tianfang; Hoekstra, Jake; Heng, Xin; Kang, Wenyan; Ding, Jianqing; Liu, Jun; Chen, Shengdi; Zhang, Jing

    2015-07-01

    Activated microglia are commonly observed in individuals with neurodegenerative disorders, including Parkinson's disease (PD) and are believed to contribute to neuronal death. This process occurs at least due partially to nicotinamide adenine dinucleotide phosphate oxidase (PHOX) activation, which leads to the production of superoxide and oxidative stress. ?-Synuclein (?-Syn), a key protein implicated in PD pathogenesis, can activate microglia, contributing to death of dopaminergic neurons. Here, microglial cells (BV2) and primary cultured microglia were used to study the role that the purinergic receptor P2X7 plays in recognizing ?-Syn and promoting PHOX activation. We demonstrate that both wild type and A53T mutant ?-Syn readily activate PHOX, with the A53T form producing more rapid and sustained effects,that is, oxidative stress and cellular injuries. Furthermore, this process involves the activation of phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway. Thus, it is concluded that stimulation of the microglial P2X7 receptor by extracellular ?-Syn, with PI3K/AKT activation and increased oxidative stress, could be an important mechanism and a potential therapeutic target for PD. PMID:25983062

  16. Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.

    PubMed Central

    Geiszt, M; Dagher, M C; Molnár, G; Havasi, A; Faure, J; Paclet, M H; Morel, F; Ligeti, E

    2001-01-01

    We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase. PMID:11311150

  17. Perturbation of actin dynamics induces NF-?B activation in myelomonocytic cells through an NADPH oxidase-dependent pathway

    PubMed Central

    2004-01-01

    Although several reports showed the effect of compounds disrupting microtubules on NF-?B (nuclear factor ?B) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-?B activation in myelomonocytic cells. The transduction pathway involved the I?B (inhibitory ?B) kinase complex and a degradation of I?B?. We have shown that NF-?B activation in response to the perturbation of actin dynamics required reactive oxygen species, as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O2? release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47phox. NF-?B activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization. PMID:15535802

  18. Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

    PubMed Central

    Grimm, Melissa J.; Lewandowski, David C.; Pham, Christine T. N.; Blackwell, Timothy S.; Petraitiene, Ruta; Petraitis, Vidmantas; Walsh, Thomas J.; Urban, Constantin F.; Segal, Brahm H.

    2011-01-01

    NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox?/?) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)?/?×cathepsin G (CG)?/? mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox?/? mice, whereas NE?/?×CG?/? mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens. PMID:22163282

  19. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, ?-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

  20. Neuronal NAD(P)H Oxidases Contribute to ROS Production and Mediate RGC Death after Ischemia

    PubMed Central

    Dvoriantchikova, Galina; Grant, Jeff; Santos, Andrea Rachelle C.; Hernandez, Eleut; Ivanov, Dmitry

    2012-01-01

    Purpose. To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase–dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. Methods. Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. Results. It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase–specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. Conclusions. The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases. PMID:22467573

  1. Targeting NADPH Oxidases for the Treatment of Cancer and Inflammation

    PubMed Central

    Bonner, Michael Y.; Arbiser, Jack L

    2015-01-01

    NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these Highly reactive and short lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in subunit composition, cellular location, and tissue specific expression. While the complexity of the nox family (Nox1–5, Duox1,2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. This review will discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of nox inhibitors. PMID:22581366

  2. NADPH Oxidase-Dependent Mechanism Explains How Arsenic and Other Oxidants Can Activate Aryl Hydrocarbon Receptor Signaling.

    PubMed

    Mohammadi-Bardbori, Afshin; Vikström Bergander, Linda; Rannug, Ulf; Rannug, Agneta

    2015-12-21

    The mechanisms explaining arsenic toxicity are not well understood, but physiological consequences of stimulated aryl hydrocarbon receptor (AHR) signaling both directly and through cross-talk with other pathways have been indicated. The aim of this study was to establish how arsenic interacts with AHR-mediated transcription. The human hepatoma cell line (HepG2-XRE-Luc) carrying a luciferase reporter under the control of two AHR response elements (AHREs) and immortalized human keratinocytes (HaCaT) were exposed to sodium arsenite (NaAsO2; As(3+)), alone or in combination with the endogenous high affinity AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Luciferase activity, cytochrome P4501A1 (CYP1A1) activity, oxidative stress-related responses, metabolic clearance of FICZ, and NADPH oxidase (NOX) activity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression were measured. Arsenic inhibited CYP1A1 enzyme activity and reduced the metabolic clearance of FICZ. Arsenic also led to activated CYP1A1 transcription but only in cells grown in medium containing trace amounts of the endogenous ligand FICZ, pointing to an indirect mechanism of activation. Initially, arsenic caused dose-dependent inhibition of FICZ-activated AHR signaling, disturbed intracellular GSH status, and increased expression of oxidative stress-related genes. Silencing of NOX4, addition of N-acetylcystein, or pretreatment with arsenic itself attenuated the initial dose-dependent inhibition of AHR signaling. Arsenic pretreatment led to elevated GSH levels and sensitized the cells to ligand-dependent AHR signaling, while silencing of Nrf2 significantly reduced arsenic-mediated activation of the AHR. In addition, influence of NOX on AHR activation was also observed in cells treated with the SH-reactive metals cadmium, mercury, and nickel. Together, the results suggest that SH-reactive agents via a new and possibly general NOX/H2O2-dependent mechanism can interfere with the endogenous regulation of the AHR. PMID:26535918

  3. NADPH oxidases are critical targets for prevention of ethanol-induced bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms through which chronic alcohol consumption induce bone loss and osteoporosis are largely unknown. Ethanol increases expression and activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzymes (Nox) in osteoblasts leading to accumulation of reactive oxygen spe...

  4. Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

    PubMed Central

    Pendyala, Srikanth; Usatyuk, Peter V.; Gorshkova, Irina A.; Garcia, Joe G.N.

    2009-01-01

    The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (Noxes and p22phox). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase–derived ROS in the pathobiology of lung diseases. Antioxid. Redox Signal. 11, 841–860. PMID:18828698

  5. Farrerol can attenuate the aortic lesion in spontaneously hypertensive rats via the upregulation of eNOS and reduction of NAD(P)H oxidase activity.

    PubMed

    Qin, Xiaojiang; Hou, Xiaomin; Liang, Taigang; Chen, Lijun; Lu, Taotao; Li, Qingshan

    2015-12-15

    Farrerol, a typical natural flavanone, is the major active component of Rhododendron dauricum L. The objective of this study was to evaluate the attenuation effect of farrerol against the aortic lesions in spontaneously hypertensive rats (SHR) for the first time. Twelve-week-old male SHR were orally administered with farrerol (50mg/kg/day), verapamil (50mg/kg/day, positive control), or vehicle for 8 weeks (n=10 in each group). Age-matched Wistar-Kyoto rats (WKY) served as normal controls (n=10). Our results revealed that farrerol significantly reduced the systolic blood pressure in SHR (from 177±4mmHg to 158±5mmHg) and also dramatically attenuated the aortic lesion, which is characterized by decreased media thickness, wall area, media-lumen ratio, nuclei size and an increased nuclei number (P<0.05). Moreover, the levels of O2(-) along with NAD(P)H oxidase activity were reduced (P<0.05), while the activity of endothelial nitric oxide synthase (eNOS) was elevated (P<0.05) in aortic homogenates after the intervention of farrerol. Furthermore, farrerol upregulated the expression of eNOS in both of mRNA and protein levels, accompanied by the downregulated mRNA and protein expression of p22(phox) (P<0.05), an essential subunit for NADPH oxidase activity. Our findings indicated that farrerol has a significant protective effect against the aortic lesion in SHR, which may be related to the enhanced eNOS activity and reduced NAD(P)H oxidase activity. PMID:26593430

  6. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone.

    PubMed Central

    Rajagopalan, S; Kurz, S; Münzel, T; Tarpey, M; Freeman, B A; Griendling, K K; Harrison, D G

    1996-01-01

    We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome-encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation. PMID:8621776

  7. ERK1/2 pathway is involved in renal gluconeogenesis inhibition under conditions of lowered NADPH oxidase activity.

    PubMed

    Winiarska, Katarzyna; Jarzyna, Robert; Dzik, Jolanta M; Jagielski, Adam K; Grabowski, Michal; Nowosielska, Agata; Focht, Dorota; Sierakowski, Bartosz

    2015-04-01

    The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has delivered some new insights into the recently discussed issue of the usefulness of Nox inhibition as a potential antidiabetic strategy. PMID:25601753

  8. NADPH oxidase mediates the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage.

    PubMed

    Tang, Xiangqi; Zhong, Wei; Tu, Qiuyun; Ding, Binrong

    2014-02-01

    Oxygen free radicals and their reactive lipid peroxidation are known to be elements promoting ischemia-reperfusion damage. NADPH oxidase is a major factor in peroxide production. Excessive production of oxygen free radicals is considered as an important mechanism in the expression of matrix metalloproteinase (MMP)-9 and in damage to the blood-brain barrier (BBB). In this study, we evaluated changes in the expression of the NADPH oxidase catalytic subunit gp91(phox) and oxidase activity, as well as the involvement of NADPH oxidase catalysis in the expression of MMP-9 in cerebral tissue after ischemia-reperfusion damage. A middle cerebral artery occlusion (MCAO) model was established using male Sprague-Dawley (SD) rats. Brain tissue was isolated for triphenyltetrazolium chloride (TTC) staining, gp91(phox) mRNA quantitative PCR analysis, western blot analysis, NADPH oxidase activity determination (detection), and MMP-9 gelatin zymography analysis. In the MCAO rats, gp91(phox) and MMP-9 expression was upregulated in the ischemic hemisphere of the brain tissue after 90 minutes of MCAO with 22·5 hours of reperfusion. Inhibition of NADPH oxidase with apocynin reduced the increase in MMP-9. These results suggest that NADPH oxidase is a major precipitating factor for the expression of MMP-9 in the ischemic brain tissue. PMID:24131725

  9. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    PubMed Central

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of ?-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce ?-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  10. DELETION MUTAGENESIS OF p22phox SUBUNIT OF FLAVOCYTOCHROME b558: IDENTIFICATION OF REGIONS CRITICAL FOR gp91phox MATURATION AND NADPH OXIDASE ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phagocyte NADPH oxidase is a multicomponent enzyme that catalyzes the transfer of electrons from NADPH to generate the superoxide radical (O2-). The importance of this enzyme in innate immunity and inflammation is illustrated by chronic granulomatous disease (CGD), a syndrome characterized by ab...

  11. Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species.

    PubMed

    Goyal, Parag; Weissmann, Norbert; Grimminger, Friedrich; Hegel, Cornelia; Bader, Lucius; Rose, Frank; Fink, Ludger; Ghofrani, Hossein A; Schermuly, Ralph T; Schmidt, Harald H H W; Seeger, Werner; Hänze, Jörg

    2004-05-15

    Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways. PMID:15110393

  12. Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.

    PubMed

    Hahn, Nynke E; Musters, René J P; Fritz, Jan M; Pagano, Patrick J; Vonk, Alexander B A; Paulus, Walter J; van Rossum, Albert C; Meischl, Christof; Niessen, Hans W M; Krijnen, Paul A J

    2014-09-01

    Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 ?M PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. PMID:24794531

  13. Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells

    PubMed Central

    Hahn, Nynke E.; Musters, René J.P.; Fritz, Jan M.; Pagano, Patrick J.; Vonk, Alexander B.A.; Paulus, Walter J.; van Rossum, Albert C.; Meischl, Christof; Niessen, Hans W.M.; Krijnen, Paul A.J.

    2015-01-01

    Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 ?M PE to induce hypertrophy after 24 and 48 h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4 h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4 h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48 h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. PMID:24794531

  14. Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells.

    PubMed

    Sorescu, D; Somers, M J; Lassègue, B; Grant, S; Harrison, D G; Griendling, K K

    2001-03-15

    Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2*- production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2*-. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2*- with DEPMPO. We confirmed that the main source of O2*- from VSMC membranes is an NAD(P)H oxidase and that the O2*- formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2*- when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC(50)'s ( approximately 10-50 microM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O2*- (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2*-. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2*- production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme. PMID:11295358

  15. EquiNox2: A new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme.

    PubMed

    Derochette, Sandrine; Serteyn, Didier; Mouithys-Mickalad, Ange; Ceusters, Justine; Deby-Dupont, Ginette; Neven, Philippe; Franck, Thierry

    2015-11-01

    Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacological tool, to determine the direct interaction of potential inhibitors with equine phagocytic NOX and their effect on the enzyme activity or assembly. This method consists in binding the membrane fractions of neutrophils containing flavocytochrome b558 or the entire complex, reconstituted in vitro from membrane and cytosolic fractions of PMNs, onto the wells of a microplate followed by incubation with potential inhibitors or drugs. After incubation, the excess of the drug is simply eliminated or washed prior measuring the activity of the reconstituted complex. This latter step avoid the risk of interference between the inhibitor and the revelation solution and can distinguish if inhibitors, strongly bound or not, could interfere with the assembly of the enzymatic complex or with its activity. The EquiNox2 was validated using diphenyliodonium chloride and Gp91ds-tat, two well-known inhibitors largely described for human NADPH oxidase. The present technique was used to study and understand better the effect of curcumin and its water-soluble derivative, NDS27, on the assembly and activity of NOX. We demonstrated that curcumin and NDS27 can strongly bind to the enzyme and prevents its assembly making these molecules good candidates for the treatment of horse or human pathologies implying an excessive activation of neutrophils. PMID:26452955

  16. Rap1 GTPase Inhibits Tumor Necrosis Factor-?-Induced Choroidal Endothelial Migration via NADPH Oxidase- and NF-?B-Dependent Activation of Rac1.

    PubMed

    Wang, Haibo; Fotheringham, Lori; Wittchen, Erika S; Hartnett, M Elizabeth

    2015-12-01

    Macrophage-derived tumor necrosis factor (TNF)-? has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-? in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-? mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-? colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-? with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-?-induced ROS generation. Apocynin reduced TNF-?-induced NF-?B and Rac1 activation, and inhibited TNF-?-induced CEC migration. TNF-?-induced Rac1 activation and CEC migration were inhibited by NF-?B inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-?-induced ROS generation and reduced NF-?B and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio)adenosine-2'-O-Me-cAMP prevented TNF-?-induced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-?B and Rac1. These findings provide evidence that active Rap1a inhibits TNF-?-induced CEC migration by inhibiting NADPH oxidase-dependent NF-?B and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROS-dependent signaling in several steps of the pathogenic process. PMID:26476350

  17. NADPH oxidase 2-derived reactive oxygen species in the hippocampus might contribute to microglial activation in postoperative cognitive dysfunction in aged mice.

    PubMed

    Qiu, Li-Li; Ji, Mu-Huo; Zhang, Hui; Yang, Jiao-Jiao; Sun, Xiao-Ru; Tang, Hui; Wang, Jing; Liu, Wen-Xue; Yang, Jian-Jun

    2016-01-01

    Microglial activation plays a key role in the development of postoperative cognitive dysfunction (POCD). Nox2, one of the main isoforms of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the central nervous system, is a predominant source of reactive oxygen species (ROS) overproduction in phagocytes including microglia. We therefore hypothesized that Nox2-induced microglial activation is involved in the development of POCD. Sixteen-month-old C57BL/6 mice were subjected to exploratory laparotomy with isoflurane anesthesia to mimic the clinical human abdominal surgery. Behavioral tests were performed at 6 and 7 d post-surgery with open field and fear conditioning tests, respectively. The levels of Nox2, 8-hydroxy-2'-deoxyguanosine (8-OH-dG, a marker of DNA oxidation), CD11b (a marker of microglial activation), interleukin-1? (IL-1?), and brain-derived neurotrophic factor (BDNF) were determined in the hippocampus and prefrontal cortex at 1 d and 7 d post-surgery, respectively. For the interventional study, mice were treated with a NADPH oxidase inhibitor apocynin (APO). Our results showed that exploratory laparotomy with isoflurane anesthesia impaired the contextual fear memory, increased expression of Nox2, 8-OH-dG, CD11b, and IL-1?, and down-regulated BDNF expression in the hippocampus at 7 d post-surgery. The surgery-induced microglial activation and neuroinflammation persisted to 7 d after surgery in the hippocampus, but only at 1 d in the prefrontal cortex. Notably, administration with APO could rescue these surgery-induced cognitive impairments and associated brain pathology. Together, our data suggested that Nox2-derived ROS in hippocampal microglia, at least in part, contributes to subsequent neuroinflammation and cognitive impairments induced by surgery in aged mice. PMID:26254234

  18. Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.

    PubMed

    Pick, E; Bromberg, Y; Shpungin, S; Gadba, R

    1987-12-01

    Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen. PMID:2824496

  19. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 (?=?ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  20. NAD(P)H oxidases and their relevance to atherosclerosis.

    PubMed

    Sorescu, D; Szöcs, K; Griendling, K K

    2001-01-01

    Studies performed during the last decade have identified NAD(P)H oxidases unique to nonphagocytic vascular cells. The reactive oxygen species released from these enzymes regulate fundamental cellular functions such as growth (hyperplastic or hypertrophic), endothelial dysfunction, migration and inflammation, which have been demonstrated to play a role in atherogenesis. Evidence from experimental animal and human studies implicate the nonphagocytic NAD(P)H oxidases in multiple aspects of atherogenesis, suggesting that these enzymes may be important determinants of the course of vascular disease. PMID:11686001

  1. Role of NADPH Oxidase and Stat3 in Statin-Mediated Protection against Diabetic Retinopathy

    PubMed Central

    Al-Shabrawey, Mohamed; Bartoli, Manuela; El-Remessy, Azza B.; Ma, Guochuan; Matragoon, Suraporn; Lemtalsi, Tahira; Caldwell, R. William; Caldwell, Ruth B.

    2010-01-01

    PURPOSE Inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (statins) reduce signs of diabetic retinopathy in diabetic patients and animals. Indirect clinical evidence supports the actions of statins in improving cardiovascular function, but the mechanisms of their protective actions in the retina are not understood. Prior studies have implicated oxidative stress and NADPH oxidase–mediated activation of signal transducer and activator of transcription 3 (STAT3) in diabetes-induced increases in expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 and breakdown of the blood–retinal barrier (BRB). Because statins are known to be potent antioxidants, the hypothesis for the current study was that the protective effects of statins in preventing diabetic retinopathy involve blockade of diabetes-induced activation of NADPH oxidase and STAT3. METHODS The hypothesis was tested by experiments in which rats with streptozotocin (STZ)-induced diabetes and retinal endothelial cells maintained in high-glucose medium were treated with simvastatin. Blood–retinal barrier (BRB) function was assayed by determining extravasation of albumin. Oxidative stress was assayed by measuring lipid peroxidation, protein nitration of tyrosine, dihydroethidine oxidation, and chemiluminescence. Immunoprobe techniques were used to determine the levels of NADPH oxidase subunit expression and STAT3 activation. RESULTS These studies showed that simvastatin blocks diabetes or high-glucose–induced increases in VEGF and ICAM-1 and preserves the BRB by a process involving blockade of diabetes/high-glucose–induced activation of STAT3 and NADPH oxidase. Statin treatment also prevents diabetes-induced increases in expression of the NADPH oxidase catalytic and subunit NOX2. CONCLUSIONS These results suggest that simvastatin protects against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of STAT3. PMID:18378570

  2. NAD(P)H oxidase: role in cardiovascular biology and disease.

    PubMed

    Griendling, K K; Sorescu, D; Ushio-Fukai, M

    2000-03-17

    Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent work has shown that NAD(P)H oxidases are major sources of superoxide in vascular cells and myocytes. The biochemical characterization, activation paradigms, structure, and function of this enzyme are now partly understood. Vascular NAD(P)H oxidases share some, but not all, characteristics of the neutrophil enzyme. In response to growth factors and cytokines, they produce superoxide, which is metabolized to hydrogen peroxide, and both of these reactive oxygen species serve as second messengers to activate multiple intracellular signaling pathways. The vascular NAD(P)H oxidases have been found to be essential in the physiological response of vascular cells, including growth, migration, and modification of the extracellular matrix. They have also been linked to hypertension and to pathological states associated with uncontrolled growth and inflammation, such as atherosclerosis. PMID:10720409

  3. Mitochondrial Regulation of NADPH Oxidase in Hindlimb Unweighting Rat Cerebral Arteries

    PubMed Central

    Xu, Fei; Sun, Jun-fang; Zhang, Lan-ning; Fan, Yong-yan; Peng, Li; Cui, Geng

    2014-01-01

    Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation. PMID:24759683

  4. iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells

    PubMed Central

    Wu, Feng; Tyml, Karel; Wilson, John X.

    2008-01-01

    Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-? (LPS+IFN?)-stimulated microvascular endothelial cells from mouse skeletal muscles. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS+IFN? caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS+IFN? induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate, NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NF?B activation. LPS+IFN? stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of I?B, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS+IFN?-induced DNA binding activity of transcription factors IRF1 and AP1 but not NF?B. In conclusion, LPS+IFN?-induced NF?B activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS+IFN?-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NF?B activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis. PMID:18481258

  5. NADPH oxidase mediates TNF-?-evoked in vitro brain barrier dysfunction: roles of apoptosis and time.

    PubMed

    Abdullah, Zuraidah; Bayraktutan, Ulvi

    2014-07-01

    The pro-inflammatory cytokine TNF-? severely perturbs the integrity of the blood-brain barrier (BBB). This study explored the specific roles of NADPH oxidase and associated downstream effectors by using human brain microvascular endothelial cells (HBMECs) and human astrocytes (HAs), the key components of BBB, alone or in co-cultures to mimic human BBB. Exposure to TNF-? (6h) impaired BBB integrity as evidenced by marked decreases in transendothelial electrical resistance and concurrent increases in paracellular flux which appeared to subside with time (24h). Increased barrier dysfunction concurred with increases in endothelial NADPH oxidase activity, O2(-) production, actin stress fibre formation, MMP-2/9 activities and concomitant decreases in antioxidant (CuZn-SOD and catalase) and tight junction (claudin-5 and occludin) protein expressions. Conversely, TNF-? did not affect astrocytic MMP activities and antioxidant enzyme expressions. Unlike BBB damage, rates of HBMEC and HA apoptosis increased by time. Suppression of NADPH oxidase by apocynin or diphenyleneiodonium prevented TNF-?-evoked morphological changes and apoptosis, attenuated endothelial MMP activity and helped retain usual tight junction protein expression and barrier function. In conclusion, HBMECs constitute the main source of oxidative stress and basement-membrane degrading endopeptidases in inflammatory conditions associated with excessive release of TNF-? where targeting NADPH oxidase may prove extremely beneficial in maintaining proper barrier activity through prevention of cytoskeletal and tight junction reorganisations. PMID:24907586

  6. Inhibition by alkylamines of NADPH oxidase through blocking the assembly of enzyme components.

    PubMed

    Sawai, T; Asada, M; Nishizawa, Y; Nunoi, H; Katayama, K

    1999-07-01

    Alkylamines inhibit NADPH oxidase both in intact neutrophils and in a cell-free system. The aim of this study was to examine the mechanism underlying this inhibitory effect. Among alkylamines with different chain lengths, the C12 compound (laurylamine) showed the greatest inhibitory effect on the cell-free NADPH oxidase activity induced by arachidonic acid (AA) in the presence of GTPgammaS. The inhibition was overcome by further addition of AA, and it was observed irrespective of whether laurylamine was added before or after the enzyme activation by AA. When added prior to the enzyme activation, laurylamine blocked translocation to the membrane of all three cytosolic components (p47-phox, p67-phox and rac) in a cell-free translocation assay. When added after the activation, laurylamine released only rac from the membrane. Laurylamine did not inhibit the reduction of cytochrome c by xanthine oxidase, suggesting that it does not have superoxide-scavenging activity. These results indicate that laurylamine inhibits both the activation process of NADPH oxidase and the activated enzyme itself by blocking the assembly of the oxidase components. PMID:10461769

  7. Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous in vitro data suggest that ethanol (EtOH) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption obse...

  8. Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum?†

    PubMed Central

    Montero-Barrientos, M.; Hermosa, R.; Cardoza, R. E.; Gutiérrez, S.; Monte, E.

    2011-01-01

    The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

  9. NADPH Oxidase 4 Induces Cardiac Arrhythmic Phenotype in Zebrafish*

    PubMed Central

    Zhang, Yixuan; Shimizu, Hirohito; Siu, Kin Lung; Mahajan, Aman; Chen, Jau-Nian; Cai, Hua

    2014-01-01

    Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked up-regulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation, which is accompanied by overproduction of reactive oxygen species (ROS). In this study, we investigated the impact on the cardiac phenotype of NOX4 overexpression in zebrafish. One-cell stage embryos were injected with NOX4 RNA prior to video recording of a GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24–31 h post-fertilization. Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean S.D. of beat-to-beat intervals was 0.027 s/beat in control embryos versus 0.038 s/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino co-injection, treatments of the embryos with polyethylene glycol-conjugated superoxide dismutase, or NOX4 inhibitors fulvene-5, 6-dimethylamino-fulvene, and proton sponge blue. Injection of NOX4-P437H mutant RNA had no effect on the cardiac phenotype or ROS production. In addition, phosphorylation of calcium/calmodulin-dependent protein kinase II was increased in NOX4 embryos but diminished by polyethylene glycol-conjugated superoxide dismutase, whereas its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies the development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia. PMID:24962575

  10. NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

    PubMed Central

    Cave, Alison; Grieve, David; Johar, Sofian; Zhang, Min; Shah, Ajay M

    2005-01-01

    Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease. PMID:16321803

  11. Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma

    PubMed Central

    Abdala-Valencia, Hiam; Earwood, Julie; Bansal, Shelly; Jansen, Michael; Babcock, George; Garvy, Beth; Wills-Karp, Marsha; Cook-Mills, Joan M.

    2009-01-01

    Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFN?, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies. PMID:17293377

  12. Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma.

    PubMed

    Abdala-Valencia, Hiam; Earwood, Julie; Bansal, Shelly; Jansen, Michael; Babcock, George; Garvy, Beth; Wills-Karp, Marsha; Cook-Mills, Joan M

    2007-05-01

    Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNgamma, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies. PMID:17293377

  13. Calcium dependent Nox5 NADPH oxidase contributes to vascular oxidative stress in human coronary artery disease

    PubMed Central

    Guzik, Tomasz J; Chen, Wei; Gongora, Maria C.; Guzik, Bartlomiej; Lob, Heinrich E.; Mangalat, Deepa; Hoch, Nyssa; Dikalov, Sergey; Rudzinski, Pawel; Kapelak, Boguslaw; Sadowski, Jerzy; Harrison, David G

    2008-01-01

    Objectives To examine the expression and activity of the calcium dependent NADPH oxidase in human atherosclerotic coronary arteries. Background The Nox based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several Nox homologs have been identified but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear. Nox5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels. Methods Coronary arteries from patients undergoing cardiac transplant with CAD or without CAD were studied. Nox5 was quantified and visualized using Western blotting, immunofluorescence and quantitative real-time PCR. Calcium dependent NADPH oxidase activity, corresponding greatly to Nox5 activity was measured by electron paramagnetic resonance. Results Both western blotting and quantitative real time PCR indicated a marked increase in Nox5 protein and mRNA in CAD vs non CAD vessels. Calcium dependent NADPH driven production of reactive oxygen species in vascular membranes, reflecting Nox5 activity was increased 7 fold in CAD and correlated significantly with Nox5 mRNA levels among subjects. Immunofluorescence demonstrated that Nox5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced in coronary lesions. Conclusions These studies identify Nox5 as a novel, calcium dependent source of reactive oxygen species in atherosclerosis. PMID:19022160

  14. Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth.

    PubMed

    Bayraktutan, Ulvi

    2004-02-01

    Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O(2)(-)) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately threefold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100 microM) increased cell proliferation as assessed via three independent methods, i.e. cell counting, determination of total cellular protein levels and [(3)H]-thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O(2)(-)) generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO(-)) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [(3)H]-thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis. PMID:14871555

  15. Reactive Oxygen Species and Angiogenesis: NADPH Oxidase as Target for Cancer Therapy

    PubMed Central

    Ushio-Fukai, Masuko; Nakamura, Yoshimasa

    2009-01-01

    Angiogenesis is essential for tumor growth, metastasis, arteriosclerosis as well as embryonic development and wound healing. Its process is dependent on cell proliferation, migration and capillary tube formation in endothelia cells (ECs). High levels of reactive oxygen species (ROS) such as superoxide and H2O2 are observed in various cancer cells. Accumulating evidence suggests that ROS function as signaling molecules to mediate various growth-related responses including angiogenesis. ROS-dependent angiogenesis can be regulated by endogenous antioxidant enzymes such as SOD and thioredoxin. Vascular endothelial growth factor (VEGF), one of the major angiogenesis factor, is induced in growing tumors and stimulates EC proliferation and migration primarily through the VEGF receptor type2 (VEGFR2, Flk1/KDR). Major source of ROS in ECs is a NADPH oxidase which consists of Nox1, Nox2, Nox4, Nox5, p22phox, p47phox and the small G protein Rac1. NADPH oxidase is activated by various growth factors including VEGF and angiopoietin-1 as well as hypoxia and ischemia, and ROS derived from this oxidase are involved in VEGFR2 autophosphorylation, and diverse redox signaling pathways leading to induction of transcription factors and genes involved in angiogenesis. Dietary antioxidants appear to be effective for treatment of tumor angiogenesis. The aim of this review is to provide an overview of the recent progress on role of ROS derived from NADPH oxidase and redox signaling events involved in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for tumor angiogenesis. PMID:18406051

  16. Enhanced ROS production and redox signaling with combined arsenite and UVA exposure: contribution of NADPH oxidase.

    PubMed

    Cooper, Karen L; Liu, Ke Jian; Hudson, Laurie G

    2009-08-15

    Solar ultraviolet radiation (UVR) is the major etiological factor in skin carcinogenesis. However, in vivo studies demonstrate that mice exposed to arsenic and UVR exhibit significantly more tumors and oxidative DNA damage than animals treated with either agent alone. Interactions between arsenite and UVR in the production of reactive oxygen species (ROS) and stress-associated signaling may provide a basis for the enhanced carcinogenicity. In this study keratinocytes were pretreated with arsenite (3 microM) and then exposed to UVA (10 kJ/m(2)). We report that exposure to UVA after arsenite pretreatment enhanced ROS production, p38 MAP kinase activation, and induction of a redox-sensitive gene product, heme oxygenase-1, compared to either stimulus alone. UVR exposure resulted in rapid and transient NADPH oxidase activation, whereas the response to arsenite was more pronounced and persistent. Inhibition of NADPH oxidase decreased ROS production in arsenite-treated cells but had little impact on UVA-exposed cells. Furthermore, arsenite-induced, but not UVA-induced, p38 activation and HO-1 expression were dependent upon NADPH oxidase activity. These findings indicate differences in the mechanisms of ROS production by arsenite and UVA that may provide an underlying basis for the observed enhancement of redox-related cellular responses upon combined UVA and arsenite exposure. PMID:19414066

  17. The NADPH-oxidase AtrbohB plays a role in Arabidopsis seed after-ripening

    E-print Network

    Leubner, Gerhard

    The NADPH-oxidase AtrbohB plays a role in Arabidopsis seed after-ripening Kerstin Mu¨ller1 , Anna), seed dormancy, seed germination, NADPH-oxidase (Rboh). Summary · Seeds can enter a state of dormancy, in which they do not germinate under optimal environmental conditions. Dormancy can be broken during seed

  18. Differential roles of NADPH oxidases in vascular physiology and pathophysiology

    PubMed Central

    Amanso, Angelica M.; Griendling, Kathy K.

    2012-01-01

    Reactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur in discrete subcellular locations, allowing for specific, compartmentalized signaling. Among the many sources of ROS in the vessel wall, NADPH oxidases are implicated in physiological functions such as control of vasomotor tone, regulation of extracellular matrix and phenotypic modulation of vascular smooth muscle cells. They are involved in the response to injury, whether as an oxygen sensor during hypoxia, as a regulator of protein processing, as an angiogenic stimulus, or as a mechanism of wound healing. These enzymes have also been linked to processes leading to disease development, including migration, proliferation, hypertrophy, apoptosis and autophagy. As a result, NADPH oxidases participate in atherogenesis, systemic and pulmonary hypertension and diabetic vascular disease. The role of ROS in each of these processes and diseases is complex, and a more full understanding of the sources, targets, cell-specific responses and counterbalancing mechanisms is critical for the rational development of future therapeutics. PMID:22202108

  19. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    PubMed Central

    Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D’Amato, Gennaro; Ederli, Luisa

    2011-01-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O3) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O3, determined from the mRNA levels of the major allergens. We conclude that O3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. PMID:21605929

  20. Reactive oxygen species, mitochondria, and NAD(P)H oxidases in the development and progression of heart failure.

    PubMed

    Sorescu, Dan; Griendling, Kathy K

    2002-01-01

    Reactive oxygen species (ROS) released acutely in large amounts have been traditionally implicated in the cell death associated with myocardial infarction or reperfusion injury. These ROS can be released from the cardiac myocyte mitochondria, xanthine oxidase, and the phagocytic nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase. Interestingly, the chronic release of ROS has been recently linked to the development of left ventricular hypertrophy and heart failure progression. The chronic release of ROS appears to derive from the nonphagocytic NAD(P)H oxidase and mitochondria. Experimental data are accumulating suggesting that the release of ROS is required for the normal, physiologic activity of cardiac cells, but abnormal activation of the nonphagocytic NAD(P)H oxidase in response to neurohormones (angiotensin II, norepinephrine, tumor necrosis factor-a) has been shown to contribute to cardiac myocyte hypertrophy. Furthermore, the fibrosis, collagen deposition, and metalloproteinase activation involved in the remodeling of the failing myocardium are dependent on ROS released during the phenotypic transformation of fibroblasts to myofibroblasts associated with progression of end-stage heart failure. Future studies are necessary to identify the sources, mechanisms of activation of NAD(P)H oxidases, and downstream signaling targets implicated in the progression of chronic heart failure. PMID:12045381

  1. Traumatic Brain Injury and NADPH Oxidase: A Deep Relationship

    PubMed Central

    Prata, Cecilia; Vieceli Dalla Sega, Francesco; Piperno, Roberto; Hrelia, Silvana

    2015-01-01

    Traumatic brain injury (TBI) represents one of the major causes of mortality and disability in the world. TBI is characterized by primary damage resulting from the mechanical forces applied to the head as a direct result of the trauma and by the subsequent secondary injury due to a complex cascade of biochemical events that eventually lead to neuronal cell death. Oxidative stress plays a pivotal role in the genesis of the delayed harmful effects contributing to permanent damage. NADPH oxidases (Nox), ubiquitary membrane multisubunit enzymes whose unique function is the production of reactive oxygen species (ROS), have been shown to be a major source of ROS in the brain and to be involved in several neurological diseases. Emerging evidence demonstrates that Nox is upregulated after TBI, suggesting Nox critical role in the onset and development of this pathology. In this review, we summarize the current evidence about the role of Nox enzymes in the pathophysiology of TBI. PMID:25918580

  2. NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

    PubMed Central

    Kuwabara, Wilson Mitsuo Tatagiba; Zhang, Liling; Schuiki, Irmgard; Curi, Rui; Volchuk, Allen; Alba-Loureiro, Tatiana Carolina

    2015-01-01

    Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells. PMID:25668518

  3. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model

    E-print Network

    Harraz, Maged M.; Marden, Jennifer J.; Zhou, Weihong; Zhang, Yulong; Williams, Aislinn; Schö neich, Christian; Engelhardt, John F.

    2008-02-01

    •–) to H2O2. Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase–dependent (Nox-dependent) O2•– production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H2O2 uncoupled SOD1...

  4. Loss of functional NADPH oxidase-2 protects against alcohol-induced bone resorption in female p47phox-/- mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In bone, oxidant signaling through NADPH oxidase (NOX)-derived reactive oxygen species (ROS) is an important stimulus for osteoclast differentiation and activity. We have previously demonstrated that chronic alcohol abuse produces bone loss through NOX-dependent mechanisms. In the current study, s...

  5. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    PubMed Central

    Hidalgo, María A.; Carretta, María D.; Teuber, Stefanie E.; Zárate, Cristian; Cárcamo, Leonardo; Concha, Ilona I.; Burgos, Rafael A.

    2015-01-01

    N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-?B pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-?B was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-?B translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-?B pathways in neutrophils. PMID:26634216

  6. NOS1 induces NADPH oxidases and impairs contraction kinetics in aged murine ventricular myocytes.

    PubMed

    Villmow, Marten; Klöckner, Udo; Heymes, Christophe; Gekle, Michael; Rueckschloss, Uwe

    2015-09-01

    Nitric oxide (NO) modulates calcium transients and contraction of cardiomyocytes. However, it is largely unknown whether NO contributes also to alterations in the contractile function of cardiomyocytes during aging. Therefore, we analyzed the putative role of nitric oxide synthases and NO for the age-related alterations of cardiomyocyte contraction. We used C57BL/6 mice, nitric oxide synthase 1 (NOS1)-deficient mice (NOS1(-/-)) and mice with cardiomyocyte-specific NOS1-overexpression to analyze contractions, calcium transients (Indo-1 fluorescence), acto-myosin ATPase activity (malachite green assay), NADPH oxidase activity (lucigenin chemiluminescence) of isolated ventricular myocytes and cardiac gene expression (Western blots, qPCR). In C57BL/6 mice, cardiac expression of NOS1 was upregulated by aging. Since we found a negative regulation of NOS1 expression by cAMP in isolated cardiomyocytes, we suggest that reduced efficacy of ?-adrenergic signaling that is evident in aged hearts promotes upregulation of NOS1. Shortening and relengthening of cardiomyocytes from aged C57BL/6 mice were decelerated, but were normalized by pharmacological inhibition of NOS1/NO. Cardiomyocytes from NOS1(-/-) mice displayed no age-related changes in contraction, calcium transients or acto-myosin ATPase activity. Aging increased cardiac expression of NADPH oxidase subunits NOX2 and NOX4 in C57BL/6 mice, but not in NOS1(-/-) mice. Similarly, cardiac expression of NOX2 and NOX4 was upregulated in a murine model with cardiomyocyte-specific overexpression of NOS1. We conclude that age-dependently upregulated NOS1, putatively via reduced efficacy of ?-adrenergic signaling, induces NADPH oxidases. By increasing nitrosative and oxidative stress, both enzyme systems act synergistically to decelerate contraction of aged cardiomyocytes. PMID:26173391

  7. NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives

    PubMed Central

    McCann, Sarah K.; Roulston, Carli L.

    2013-01-01

    Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation. PMID:24961415

  8. Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane.

    PubMed

    Dupuy, C; Virion, A; Ohayon, R; Kaniewski, J; Dème, D; Pommier, J

    1991-02-25

    The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-. PMID:1995628

  9. Francisella Acid Phosphatases Inactivate the NADPH Oxidase in Human Phagocytes

    PubMed Central

    Mohapatra, Nrusingh P.; Soni, Shilpa; Rajaram, Murugesan V. S.; Dang, Pham My-Chan; Reilly, Tom J.; El-Benna, Jamel; Clay, Corey D.; Schlesinger, Larry S.; Gunn, John S.

    2010-01-01

    Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [?ABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the ?ABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida ?ABCH strain stimulated a significant level of reactive oxygen species. The ?ABCH mutant, but not the wild-type strain, strongly colocalized with p47phox and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47phox knockout mice. Finally, purified AcpA strongly dephosphorylated p47phox and p40phox, but not p67phox, in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes. PMID:20348422

  10. NO-mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells

    PubMed Central

    Duerrschmidt, Nicole; Stielow, Claudia; Muller, Gregor; Pagano, Patrick J; Morawietz, Henning

    2006-01-01

    The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O2?) formation. In this study, we analysed the effect of laminar shear stress on ·O2? formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced ·O2? formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm?2, 24 h) down-regulated ·O2? formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91phox and p47phox. In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of ·O2? formation, gp91phox and p47phox expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates ·O2? formation, gp91phox and p47phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O2? formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91phox/p47phox-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O2? balance and the vasoprotective potential of physiological levels of laminar shear stress. PMID:16873416

  11. Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

    PubMed Central

    Yu, XiaoTong; Wang, PengYan; Shi, ZhengMing; Dong, Kun; Feng, Ping; Wang, HongXia; Wang, XueJiang

    2015-01-01

    Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC. PMID:26658815

  12. NADPH oxidases: an overview from structure to innate immunity-associated pathologies

    PubMed Central

    Panday, Arvind; Sahoo, Malaya K; Osorio, Diana; Batra, Sanjay

    2015-01-01

    Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), play important roles in immunity, cell growth, and cell signaling. In excess, however, ROS are lethal to cells, and the overproduction of these molecules leads to a myriad of devastating diseases. The key producers of ROS in many cells are the NOX family of NADPH oxidases, of which there are seven members, with various tissue distributions and activation mechanisms. NADPH oxidase is a multisubunit enzyme comprising membrane and cytosolic components, which actively communicate during the host responses to a wide variety of stimuli, including viral and bacterial infections. This enzymatic complex has been implicated in many functions ranging from host defense to cellular signaling and the regulation of gene expression. NOX deficiency might lead to immunosuppression, while the intracellular accumulation of ROS results in the inhibition of viral propagation and apoptosis. However, excess ROS production causes cellular stress, leading to various lethal diseases, including autoimmune diseases and cancer. During the later stages of injury, NOX promotes tissue repair through the induction of angiogenesis and cell proliferation. Therefore, a complete understanding of the function of NOX is important to direct the role of this enzyme towards host defense and tissue repair or increase resistance to stress in a timely and disease-specific manner. PMID:25263488

  13. NADPH oxidases: an overview from structure to innate immunity-associated pathologies.

    PubMed

    Panday, Arvind; Sahoo, Malaya K; Osorio, Diana; Batra, Sanjay

    2015-01-01

    Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), play important roles in immunity, cell growth, and cell signaling. In excess, however, ROS are lethal to cells, and the overproduction of these molecules leads to a myriad of devastating diseases. The key producers of ROS in many cells are the NOX family of NADPH oxidases, of which there are seven members, with various tissue distributions and activation mechanisms. NADPH oxidase is a multisubunit enzyme comprising membrane and cytosolic components, which actively communicate during the host responses to a wide variety of stimuli, including viral and bacterial infections. This enzymatic complex has been implicated in many functions ranging from host defense to cellular signaling and the regulation of gene expression. NOX deficiency might lead to immunosuppression, while the intracellular accumulation of ROS results in the inhibition of viral propagation and apoptosis. However, excess ROS production causes cellular stress, leading to various lethal diseases, including autoimmune diseases and cancer. During the later stages of injury, NOX promotes tissue repair through the induction of angiogenesis and cell proliferation. Therefore, a complete understanding of the function of NOX is important to direct the role of this enzyme towards host defense and tissue repair or increase resistance to stress in a timely and disease-specific manner. PMID:25263488

  14. Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

    PubMed Central

    Hirano, Kazufumi; Chen, Woei Shin; Chueng, Adeline L.W.; Dunne, Angela A.; Seredenina, Tamara; Filippova, Aleksandra; Ramachandran, Sumitra; Bridges, Angela; Chaudry, Laiq; Pettman, Gary; Allan, Craig; Duncan, Sarah; Lee, Kiew Ching; Lim, Jean; Ma, May Thu; Ong, Agnes B.; Ye, Nicole Y.; Nasir, Shabina; Mulyanidewi, Sri; Aw, Chiu Cheong; Oon, Pamela P.; Liao, Shihua; Li, Dizheng; Johns, Douglas G.; Miller, Neil D.; Davies, Ceri H.; Browne, Edward R.; Matsuoka, Yasuji; Chen, Deborah W.; Jaquet, Vincent

    2015-01-01

    Abstract Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358–374. PMID:26135714

  15. NADPH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signaling.

    PubMed

    Al-Sabbagh, Marwa; Fusi, Luca; Higham, Jenny; Lee, Yun; Lei, Kaiyu; Hanyaloglu, Aylin C; Lam, Eric W-F; Christian, Mark; Brosens, Jan J

    2011-02-01

    Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-?, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy. PMID:21159852

  16. Tumor Necrosis Factor-?-Induced Colitis Increases NADPH Oxidase 1 Expression, Oxidative Stress, and Neutrophil Recruitment in the Colon: Preventive Effect of Apocynin

    PubMed Central

    Mouzaoui, Souad; Djerdjouri, Bahia; Makhezer, Nesrine; Kroviarski, Yolande; El-Benna, Jamel; Dang, Pham My-Chan

    2014-01-01

    Reactive oxygen species- (ROS-) mediated injury has been implicated in several inflammatory disorders, including inflammatory bowel disease (IBD). NADPH oxidases (NOXs) are the major source of endogenous ROS. Here, we investigated the role of NOXs derived-ROS in a mouse model of colitis induced by the proinflammatory cytokine, tumor necrosis factor-? (TNF-?). Intraperitoneal injection of TNF? (10??g · kg?1) induced an acute inflammation of the colon and a marked increase in expression of NADPH oxidase 1 (NOX1), a colon specific NADPH oxidase isoform. TNF?-induced colitis was also characterized by high production of keratinocyte-derived chemokine (KC) and mucosal infiltration of neutrophils, NOX2-expressing cells. Concomitantly, ROS production and lipid peroxidation were significantly enhanced while catalase activity and glutathione level were reduced indicating a redox imbalance in the colon. Furthermore, the redox-sensitive MAP kinases, ERK1/2 and p38 MAPK, were activated during TNF?-induced colitis. Pretreatment of mice with apocynin, an NADPH oxidase inhibitor with antioxidant properties, before TNF? challenge, prevented all these events. These data suggest that ROS derived from NADPH oxidases (mainly NOX1 and NOX2) and MAP kinase pathways could contribute to the induction and expansion of oxidative lesions characteristics of IBD and that apocynin could potentially be beneficial in IBD treatment. PMID:25276054

  17. FFA-induced hepatic insulin resistance in vivo is mediated by PKC?, NADPH oxidase, and oxidative stress.

    PubMed

    Pereira, Sandra; Park, Edward; Mori, Yusaku; Haber, C Andrew; Han, Ping; Uchida, Toyoyoshi; Stavar, Laura; Oprescu, Andrei I; Koulajian, Khajag; Ivovic, Alexander; Yu, Zhiwen; Li, Deling; Bowman, Thomas A; Dewald, Jay; El-Benna, Jamel; Brindley, David N; Gutierrez-Juarez, Roger; Lam, Tony K T; Najjar, Sonia M; McKay, Robert A; Bhanot, Sanjay; Fantus, I George; Giacca, Adria

    2014-07-01

    Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKC? and oxidative stress play causal roles in this process and whether this occurs via IKK?- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased I?B? content, increased JNK phosphorylation (markers of IKK? and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKC? activation. Furthermore, an antisense oligonucleotide against PKC? prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKC?, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA ? PKC? ? NADPH oxidase and oxidative stress ? IKK?/JNK ? impaired hepatic insulin signaling. PMID:24824652

  18. Potential role of NADPH oxidase in pathogenesis of pancreatitis

    PubMed Central

    Cao, Wei-Li; Xiang, Xiao-Hui; Chen, Kai; Xu, Wei; Xia, Shi-Hai

    2014-01-01

    Studies have demonstrated that reactive oxygen species (ROS) are closely related to inflammatory disorders. Nicotinamide adenine dinucleotide phosphate oxidase (NOX), originally found in phagocytes, is the main source of ROS in nonphagocytic cells. Besides directly producing the detrimental highly reactive ROS to act on biomolecules (lipids, proteins, and nucleic acids), NOX can also activate multiple signal transduction pathways, which regulate cell growth, proliferation, differentiation and apoptosis by producing ROS. Recently, research on pancreatic NOX is no longer limited to inflammatory cells, but extends to the aspect of pancreatic acinar cells and pancreatic stellate cells, which are considered to be potentially associated with pancreatitis. In this review, we summarize the literature on NOX protein structure, activation, function and its role in the pathogenesis of pancreatitis. PMID:25133019

  19. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B.

    PubMed

    Park, Hye Sun; Jung, Hye Young; Park, Eun Young; Kim, Jaesang; Lee, Won Jae; Bae, Yun Soo

    2004-09-15

    LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-kappaB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-kappaB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-kappaB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-kappaB activation. PMID:15356101

  20. Endotoxin-induced vascular endothelial cell migration is dependent on TLR4/NF-?B pathway, NAD(P)H oxidase activation, and transient receptor potential melastatin 7 calcium channel activity.

    PubMed

    Sarmiento, Daniela; Montorfano, Ignacio; Cáceres, Mónica; Echeverría, César; Fernández, Ricardo; Cabello-Verrugio, Claudio; Cerda, Oscar; Tapia, Pablo; Simon, Felipe

    2014-10-01

    Endothelial dysfunction is decisive and leads to the development of several inflammatory diseases. Endotoxemia-derived sepsis syndrome exhibits a broad inflammation-induced endothelial dysfunction. We reported previously that the endotoxin, lipopolysaccharide (LPS), induces the conversion of endothelial cells (ECs) into activated fibroblasts, showing a myofibroblast-like protein expression profile. Enhanced migration is a hallmark of myofibroblast function. However, the mechanism involved in LPS-induced EC migration is no totally understood. Some studies have shown that the transient receptor potential melastatin 7 (TRPM7) ion channel is involved in fibroblast and tumor cell migration through the regulation of calcium influx. Furthermore, LPS modulates TRPM7 expression. However, whether TRPM7 is involved in LPS-induced EC migration remains unknown. Here, we study the participation of LPS as an inducer of EC migration and study the mechanism underlying evaluating the participation of the TRPM7 ion channel. Our results demonstrate that LPS induced EC migration in a dose-dependent manner. Furthermore, this migratory process was mediated by the TLR-4/NF-?B pathway and the generation of ROS through the PKC-activated NAD(P)H oxidase. In addition, LPS increased the intracellular calcium level and the number of focal adhesion kinase (FAK)-positive focal adhesions in EC. Finally, we demonstrate that using TRPM7 blockers or suppressing TRPM7 expression through siRNA successfully inhibits the calcium influx and the LPS-induced EC migration. These results point out TRPM7 as a new target in the drug design for several inflammatory diseases that impair vascular endothelium function. PMID:25130439

  1. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

    SciTech Connect

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative I?B-? plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-?B, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-?B pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to ethanol-induced MMP-12 expression. • p38 MAPK/NF-?B signaling pathway modulates ethanol-induced Nox2 expression.

  2. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    PubMed Central

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Federman Gross, Aya; Rafalowski, Meirav; Pick, Edgar

    2015-01-01

    The superoxide (O·?2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O·?2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components. PMID:25699251

  3. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 (phox.).

    PubMed

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Federman Gross, Aya; Rafalowski, Meirav; Pick, Edgar

    2015-01-01

    The superoxide (O(·-) 2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b 558 (a heterodimer of Nox2 and p22 (phox) ), and four cytosolic components, p47 (phox) , p67 (phox) , p40 (phox) , and Rac. The catalytic component, responsible for O(·-) 2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67 (phox) . Using a peptide-protein binding assay, we found that Nox2 peptides containing a (369)CysGlyCys(371) triad (CGC) bound p67 (phox) with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67 (phox) only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67 (phox) via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: (1) Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; (2) Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; (3) Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; (4) Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; (5) A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; (6) p67 (phox) , in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67 (phox) to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components. PMID:25699251

  4. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    NASA Astrophysics Data System (ADS)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  5. Alcohol-induced bone loss is blocked in p47phox -/- mice lacking functional nadph oxidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a resu...

  6. NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice.

    PubMed

    Murray, Thomas V A; Dong, Xuebin; Sawyer, Greta J; Caldwell, Anna; Halket, John; Sherwood, Roy; Quaglia, Alberto; Dew, Tracy; Anilkumar, Narayana; Burr, Simon; Mistry, Rajesh K; Martin, Daniel; Schröder, Katrin; Brandes, Ralf P; Hughes, Robin D; Shah, Ajay M; Brewer, Alison C

    2015-12-01

    Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins. PMID:26472193

  7. The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

    SciTech Connect

    Riganti, Chiara

    2008-05-01

    We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

  8. Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-?B-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1?-exposed Caco-2 cells.

    PubMed

    Tesoriere, L; Attanzio, A; Allegra, M; Gentile, C; Livrea, M A

    2014-02-01

    Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1?, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1? brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-?B, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 ?M), and IL-1? prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1? also prevented the IL-1?-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-?B was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level. PMID:23931157

  9. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

    NASA Technical Reports Server (NTRS)

    McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

    2003-01-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  10. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase

    PubMed Central

    Idelman, Gila; Smith, Darcey L.H.; Zucker, Stephen D.

    2015-01-01

    It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O2?) production, respectively. The generation of both nitrate and O2? in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated O2? production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1? (HIF-1?), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1?-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1? mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-?, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1? through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-? release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin. PMID:26163808

  11. Endothelin-1 impairs coronary arteriolar dilation: Role of p38 kinase-mediated superoxide production from NADPH oxidase.

    PubMed

    Thengchaisri, Naris; Hein, Travis W; Ren, Yi; Kuo, Lih

    2015-09-01

    Elevated levels of endothelin-1 (ET-1), a potent vasoactive peptide, are implicated as a risk factor for cardiovascular diseases by exerting vasoconstriction. The aim of this study was to address whether ET-1, at sub-vasomotor concentrations, elicits adverse effects on coronary microvascular function. Porcine coronary arterioles (50-100?m) were isolated, cannulated and pressurized without flow for in vitro study. Diameter changes were recorded using a videomicrometer. Arterioles developed basal tone (60±3?m) and dilated to the endothelium-dependent nitric oxide (NO)-mediated vasodilators serotonin (1nmol/L to 0.1?mol/L) and adenosine (1nmol/L to 10?mol/L). Treating the vessels with a clinically relevant sub-vasomotor concentration of ET-1 (10pmol/L, 60min) significantly attenuated arteriolar dilations to adenosine and serotonin but not to endothelium-independent vasodilator sodium nitroprusside. The arteriolar wall contains ETA receptors and the adverse effect of ET-1 was prevented by ETA receptor antagonist BQ123, the superoxide scavenger Tempol, the NADPH oxidase inhibitors apocynin and VAS2870, the NOX2-based NADPH oxidase inhibitor gp91 ds-tat, or the p38 kinase inhibitor SB203580. However, ETB receptor antagonist BQ788, H2O2 scavenger catalase, scrambled gp91 ds-tat, or inhibitors of xanthine oxidase (allopurinol), PKC (Gö 6983), Rho kinase (Y27632), and c-Jun N-terminal kinase (SP600125) did not protect the vessel. Immunohistochemical staining showed that ET-1 elicited Tempol-, apocynin- and SB203580-sensitive superoxide productions in the arteriolar wall. Our results indicate that exposure of coronary arterioles to a pathophysiological, sub-vasomotor concentration of ET-1 leads to vascular dysfunction by impairing endothelium-dependent NO-mediated dilation via p38 kinase-mediated production of superoxide from NADPH oxidase following ETA receptor activation. PMID:26211713

  12. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  13. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase.

    PubMed

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  14. Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways.

    PubMed

    Cui, Yanji; Park, Jee-Yun; Wu, Jinji; Lee, Ji Hyung; Yang, Yoon-Sil; Kang, Moon-Seok; Jung, Sung-Cherl; Park, Joo Min; Yoo, Eun-Sook; Kim, Seong-Ho; Ahn Jo, Sangmee; Suk, Kyoungho; Eun, Su-Yong

    2015-05-01

    Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91 (phox) , which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways. PMID:25954126

  15. Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways

    PubMed Central

    Cui, Yanji; Park, Jee-Yun; Wu, Jinji; Lee, Ji Hyung; Yang, Yoon-Sil; Kang, Moon-Seok; Jung, Sung-Cherl; Park, Joo Min; Yoo, Eun-Sook; Kim, Seong-Ho; Ahn Jo, Sangmee; Suk, Kyoungho

    2015-01-01

    Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91phox, which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways. PMID:25954126

  16. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury.

    PubMed

    Szöcs, Katalin; Lassègue, Bernard; Sorescu, Dan; Hilenski, Lula L; Valppu, Liisa; Couse, Tracey L; Wilcox, Josiah N; Quinn, Mark T; Lambeth, J David; Griendling, Kathy K

    2002-01-01

    Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O2*(-)) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O2*(-) that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O2*(-) production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7- and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis. PMID:11788456

  17. Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

    PubMed Central

    Gorin, Yves; Wauquier, Fabien

    2015-01-01

    Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-?. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted. PMID:25824546

  18. NADPH Oxidase 4 Mediates TGF?1-induced CCN2 in Gingival Fibroblasts.

    PubMed

    Yang, W H; Deng, Y T; Hsieh, Y P; Wu, K J; Kuo, M Y P

    2015-07-01

    Transforming growth factor ? (TGF?) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGF? in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGF?. We previously showed that Src, JNK, and Smad3 mediate TGF?1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGF?. In this study, we demonstrated that TGF?1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGF?1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGF?1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGF?1-induced NOX4 expression in HGFs. PMID:25858818

  19. Methamphetamine alters occludin expression via NADPH oxidase-induced oxidative insult and intact caveolae

    PubMed Central

    Park, Minseon; Hennig, Bernhard; Toborek, Michal

    2012-01-01

    Abstract Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. This study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells. PMID:21435178

  20. Fetal–maternal interface impedance parallels local NADPH oxidase related superoxide production

    PubMed Central

    Guedes-Martins, L.; Silva, E.; Gaio, A.R.; Saraiva, J.; Soares, A.I.; Afonso, J.; Macedo, F.; Almeida, H.

    2015-01-01

    Blood flow assessment employing Doppler techniques is a useful procedure in pregnancy evaluation, as it may predict pregnancy disorders coursing with increased uterine vascular impedance, as pre-eclampsia. While the local causes are unknown, emphasis has been put on reactive oxygen species (ROS) excessive production. As NADPH oxidase (NOX) is a ROS generator, it is hypothesized that combining Doppler assessment with NOX activity might provide useful knowledge on placental bed disorders underlying mechanisms. A prospective longitudinal study was performed in 19 normal course, singleton pregnancies. Fetal aortic isthmus (AoI) and maternal uterine arteries (UtA) pulsatility index (PI) were recorded at two time points: 20–22 and 40–41 weeks, just before elective Cesarean section. In addition, placenta and placental bed biopsies were performed immediately after fetal extraction. NOX activity was evaluated using a dihydroethidium-based fluorescence method and associations to PI values were studied with Spearman correlations. A clustering of pregnancies coursing with higher and lower PI values was shown, which correlated strongly with placental bed NOX activity, but less consistently with placental tissue. The study provides evidence favoring that placental bed NOX activity parallels UtA PI enhancement and suggests that an excess in oxidation underlies the development of pregnancy disorders coursing with enhanced UtA impedance. PMID:25912167

  1. Leptin regulates cardiomyocyte contractile function through endothelin-1 receptor-NADPH oxidase pathway.

    PubMed

    Dong, Feng; Zhang, Xiaochun; Ren, Jun

    2006-02-01

    Leptin, the obese gene product, plays an important role in the regulation of cardiac function. However, the mechanism behind leptin-induced cardiomyocyte contractile response is poorly understood. This study was designed to examine whether endothelin-1 receptor and NADPH oxidase play any role in leptin-induced cardiac contractile response. Isolated murine cardiomyocytes were exposed to leptin (5, 50, and 100 nmol/L) for 60 minutes in the absence or presence of the ETA receptor antagonist BQ123 (1 micromol/L), the ETB receptor antagonist BQ788 (1 micromol/L), or the NADPH oxidase inhibitor apocynin (100 micromol/L) before mechanical function was studied. Superoxide levels were measured by dihydroethidium fluorescent dye and the superoxide dismutase-inhibitable reduction of cytochrome c. NADPH oxidase subunit expression (p22phox, p47phox, p67phox, and gp91phox) was evaluated with Western blot. Leptin depressed peak shortening and maximal velocity of shortening/relengthening (+/-dL/dt), prolonged the duration of relengthening (TR90) without affecting the time-to-peak cell shortening. Consistent with the mechanical characteristics, myocytes treated with leptin displayed a reduced electrically stimulated rise in intracellular Ca2+ (change in fura-2 fluorescence intensity) associated with a prolonged intracellular Ca2+ decay rate. All of the abnormalities were significantly attenuated by apocynin, BQ123, or BQ788. Intracellular superoxide generation was enhanced after leptin treatment, which was partially blocked by apocynin, BQ123, or BQ788. Leptin had no effect on p22phox and gp91phox but upregulated protein expression of p67phox and p47phox, both of which were inhibited by apocynin, BQ123, or BQ788. These results suggest that leptin suppresses cardiac contractile function in ventricular myocytes through the endothelin-1 receptor and NADPH oxidase-mediated pathway. PMID:16380530

  2. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

    PubMed Central

    Altenhöfer, Sebastian; Radermacher, Kim A.; Kleikers, Pamela W.M.; Wingler, Kirstin

    2015-01-01

    Abstract Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406–427. PMID:24383718

  3. NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes

    PubMed Central

    Persson, Patrik; Hansell, Peter

    2012-01-01

    Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po2) due to increased oxygen consumption (Qo2). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo2 by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na+ transport and kidney Po2 in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na+ excretion, fractional Li+ excretion, and intrarenal Po2 was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 ± 0.3 vs. 1.4 ± 0.1 ml·min?1·kidney?1). RBF was similar in both groups, resulting in increased FF in diabetics. Po2 was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 ± 0.7 vs. 42.5 ± 1.2 mmHg and 15.7 ± 1.2 vs. 25.5 ± 2.3 mmHg, respectively). Na+ excretion was increased in diabetics compared with controls (24.0 ± 4.7 vs. 9.0 ± 2.0 ?m·min?1·kidney?1). In controls, all parameters were unaffected. However, apocynin increased Na+ excretion (+112%) and decreased fractional lithium reabsorption (?10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po2. Qo2 was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo2, but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na+ transport and improves intrarenal Po2 in diabetes. PMID:22552796

  4. The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea.

    PubMed

    Giesbert, Sabine; Schürg, Timo; Scheele, Sandra; Tudzynski, Paul

    2008-05-01

    The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus. PMID:18705873

  5. Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF??B pathway under high glucose conditions.

    PubMed

    Sun, Li; Li, Weiping; Li, Weizu; Xiong, Li; Li, Guiping; Ma, Rong

    2014-07-01

    Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy (DN), which are respectively related to mesangial cell (MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel 6 (TRPC6). The marked increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of ROS production in MCs. Astragaloside IV (AS?IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose (HG)?induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS?IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS?IV for 48 h markedly attenuated HG?induced proliferation and the hypertrophy of MCs in a dose?dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS?IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase 4 (Nox4) protein were decreased. Treatment with AS?IV also inhibited the phosphorylation level of Akt and I?B? in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS?IV under HG conditions. These results suggest that AS?IV inhibits HG?induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor??B (NF??B) pathway, providing a new perspective for the clinical treatment of DN. PMID:24718766

  6. Role of NADPH oxidases in the redox biology of liver fibrosis.

    PubMed

    Crosas-Molist, Eva; Fabregat, Isabel

    2015-12-01

    Liver fibrosis is the pathological consequence of chronic liver diseases, where an excessive deposition of extracellular matrix (ECM) proteins occurs, concomitantly with the processes of repair and regeneration. It is characterized by increased production of matrix proteins, in particular collagens, and decreased matrix remodelling. The principal source of ECM accumulation is myofibroblasts (MFB). Most fibrogenic MFB are endogenous to the liver, coming from hepatic stellate cells (HSC) and portal fibroblasts. Dysregulated inflammatory responses have been associated with most (if not all) hepatotoxic insults and chronic oxidative stress play a role during the initial liver inflammatory phase and its progression to fibrosis. Redox-regulated processes are responsible for activation of HSC to MFB, as well as maintenance of the MFB function. Increased oxidative stress also induces hepatocyte apoptosis, which contributes to increase the liver injury and to transdifferentiate HSC to MFB, favouring the fibrogenic process. Mitochondria and other redox-active enzymes can generate superoxide and hydrogen peroxide as a by-product in liver cells. Moreover, accumulating evidence indicates that NADPH oxidases (NOXs), which play a critical role in the inflammatory response, may contribute to reactive oxygen species (ROS) production during liver fibrosis, being important players in HSC activation and hepatocyte apoptosis. Based on the knowledge of the pathogenic role of ROS, different strategies to prevent or reverse the oxidative damage have been developed to be used as therapeutic tools in liver fibrosis. This review will update all these concepts, highlighting the relevance of redox biology in chronic fibrogenic liver pathologies. PMID:26204504

  7. Endothelin-1 Regulates Cardiac L-Type Calcium Channels via NAD(P)H Oxidase-Derived Superoxide

    PubMed Central

    Zeng, Qinghua; Zhou, Qingwei; Yao, Fanrong; O’Rourke, Stephen T.; Sun, Chengwen

    2015-01-01

    It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ETA receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 ?M)] but was not altered in the presence of an ETB receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 ?M)]. Pre-treatment of cells with the ROS scavenger tempol (100 ?M), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH3]) (5 ?M) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca2+ channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes. PMID:18539650

  8. NOX4 NADPH Oxidase-Dependent Mitochondrial Oxidative Stress in Aging-Associated Cardiovascular Disease

    PubMed Central

    Vendrov, Aleksandr E.; Vendrov, Kimberly C.; Smith, Alberto; Yuan, Jinling; Sumida, Arihiro; Robidoux, Jacques; Madamanchi, Nageswara R.

    2015-01-01

    Abstract Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe?/? mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe?/? and Apoe?/?/p47phox?/? mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox?/? mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe?/? mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD. Antioxid. Redox Signal. 23, 1389–1409. PMID:26054376

  9. Hydrogen peroxide generated by NADPH oxidase is involved in high blue-light-induced chloroplast avoidance movements in Arabidopsis

    NASA Astrophysics Data System (ADS)

    Wen, Feng; Xing, Da; Zhang, Lingrui

    2009-08-01

    One of the most important functions of blue light is to induce chloroplast movements by reducing the damage to photosynthetic machinery under excess light. Hydrogen peroxide (H2O2), generated by various environmental stimuli, can act as a signaling molecule that regulates a number of developmental processes and environmental responses. To investigate whether H2O2 is involved in high blue light-induced chloroplast avoidance movements, we use luminescence spectrometer to observe H2O2 generation with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). After treatment with high blue light, a large quantity of H2O2 indicated by the fluorescence intensity of DCF is produced in a dose-dependent manner in leaf strip of Arabidopsis. Enzymatic assay shows that the activity of NADPH oxidase, which is a major site for H2O2 generation, also rapidly increases in treated strips. Exogenously applied H2O2 can promote the high blue light-induced chloroplast movements. Moreover, high blue light-induced H2O2 generation can be abolished completely by addition of exogenous catalase (CAT), and partly by diphenylene iodonium (DPI) and dichlorophenyl dimethylurea (DCMU), which are an NADPH oxidase inhibitor and a blocker of electron transport chain. And subsequent chloroplast movements can be abolished by CAT and DPI, but not by DCMU. These results presented here suggested that high blue light can induce oxidative burst, and NADPH oxidase as a major producer for H2O2 is involved in blue light-induced chloroplast avoidance movements.

  10. Phagocyte NADPH oxidase restrains the inflammasome in ANCA-induced GN.

    PubMed

    Schreiber, Adrian; Luft, Friedrich C; Kettritz, Ralph

    2015-02-01

    ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1? generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91(phox)-deficient or p47(phox)-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1? levels compared with mice transplanted with wild-type bone marrow. IL-1? receptor blockade abrogated aggravated NCGN in gp91(phox)-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1? generation in gp91(phox)-deficient and p47(phox)-deficient monocytes compared with wild-type monocytes. This enhanced IL-1? generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1? generation were inversely related in human monocytes. Furthermore, transplantation of gp91(phox)/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91(phox) bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants. PMID:25012177

  11. Silica particles cause NADPH oxidase–independent ROS generation and transient phagolysosomal leakage

    PubMed Central

    Joshi, Gaurav N.; Goetjen, Alexandra M.; Knecht, David A.

    2015-01-01

    Chronic inhalation of silica particles causes lung fibrosis and silicosis. Silica taken up by alveolar macrophages causes phagolysosomal membrane damage and leakage of lysosomal material into the cytoplasm to initiate apoptosis. We investigated the role of reactive oxygen species (ROS) in this membrane damage by studying the spatiotemporal generation of ROS. In macrophages, ROS generated by NADPH oxidase 2 (NOX2) was detected in phagolysosomes containing either silica particles or nontoxic latex particles. ROS was only detected in the cytoplasm of cells treated with silica and appeared in parallel with an increase in phagosomal ROS, as well as several hours later associated with mitochondrial production of ROS late in apoptosis. Pharmacological inhibition of NOX activity did not prevent silica-induced phagolysosomal leakage but delayed it. In Cos7 cells, which do not express NOX2, ROS was detected in silica-containing phagolysosomes that leaked. ROS was not detected in phagolysosomes containing latex particles. Leakage of silica-containing phagolysosomes in both cell types was transient, and after resealing of the membrane, endolysosomal fusion continued. These results demonstrate that silica particles can generate phagosomal ROS independent of NOX activity, and we propose that this silica-generated ROS can cause phagolysosomal leakage to initiate apoptosis. PMID:26202463

  12. Nox Family NADPH Oxidases in Mechano-Transduction: Mechanisms and Consequences

    PubMed Central

    Weissmann, Norbert; Schröder, Katrin

    2014-01-01

    Abstract Significance: The majority of cells in a multi-cellular organism are continuously exposed to ever-changing physical forces. Mechano-transduction links these events to appropriate reactions of the cells involving stimulation of signaling cascades, reorganization of the cytoskeleton and alteration of gene expression. Recent Advances: Mechano-transduction alters the cellular redox balance and the formation of reactive oxygen species (ROS). Nicotine amide adenine dinucleotide reduced form (NADPH) oxidases of the Nox family are prominent ROS generators and thus, contribute to this stress-induced ROS formation. Critical Issues: Different types and patterns of mechano-stress lead to Nox-dependent ROS formation and Nox-mediated ROS formation contributes to cellular responses and adaptation to physical forces. Thereby, Nox enzymes can mediate vascular protection during physiological mechano-stress. Despite this, over-activation and induction of Nox enzymes and a subsequent substantial increase in ROS formation also promotes oxidative stress in pathological situations like disturbed blood flow or extensive stretch. Future Directions: Individual protein targets of Nox-mediated redox-signaling will be identified to better understand the specificity of Nox-dependent ROS signaling in mechano-transduction. Nox-inhibitors will be tested to reduce cellular activation in response to mechano-stimuli. Antioxid. Redox Signal. 20, 887–898. PMID:23682993

  13. SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

    PubMed Central

    Douda, David Nobuhiro; Khan, Meraj A.; Grasemann, Hartmut; Palaniyar, Nades

    2015-01-01

    Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed “NETosis” is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis. PMID:25730848

  14. Protection against LPS-induced acute lung injury by a mechanism-based inhibitor of NADPH oxidase (type 2)

    PubMed Central

    Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Feinstein, Sheldon I.

    2014-01-01

    The phospholipase A2 activity of peroxiredoxin 6 is inhibited by the transition state analog, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33). This activity is required for the activation of NADPH oxidase, type 2. The present study evaluated the effect of MJ33 on manifestations of acute lung injury. Mice were injected intratracheally (IT) with LPS from Escherichia coli 0111:B4 (LPS, 1 or 5 mg/kg), either concurrently with LPS or 2 h later, and evaluated for lung injury 24 h later. MJ33 inhibited reactive oxygen species (ROS) generation by lungs when measured at 24 h after LPS. LPS at either a low or high dose significantly increased lung infiltration with inflammatory cells, secretion of proinflammatory cytokines (IL-6, TNF-?, and the chemokine macrophage inflammatory protein-2), expression of lung vascular cell adhesion molecule, lung permeability (protein in bronchoalveolar lavage fluid, leakage of FITC-dextran, lung wet-to-dry weight ratio), tissue lipid peroxidation (thiobarbituric acid reactive substances, 8-isoprostanes), tissue protein oxidation (protein carbonyls), and activation of NF-?B. MJ33, given either concurrently or 2 h subsequent to LPS, significantly reduced all of these measured parameters. Previous studies of toxicity showed a high margin of safety for MJ33 in the intact mouse. Thus we have identified MJ33 as a potent, nontoxic, and specific mechanism-based inhibitor of NADPH oxidase type 2-mediated ROS generation that protects mice against lung injury associated with inflammation. PMID:24487388

  15. NADPH oxidases-do they play a role in TRPC regulation under hypoxia?

    PubMed

    Malczyk, Monika; Veith, Christine; Schermuly, Ralph T; Gudermann, Thomas; Dietrich, Alexander; Sommer, Natascha; Weissmann, Norbert; Pak, Oleg

    2016-01-01

    In the lung, acute alveolar hypoxia causes hypoxic pulmonary vasoconstriction (HPV) to maintain ventilation perfusion matching and thus optimal oxygenation of blood. In contrast, global chronic hypoxia triggers a pathological thickening of pulmonary arterial walls, called pulmonary vascular remodelling, leading to persistence of pulmonary hypertension (PH). Moreover, ischaemia or hypoxia can lead to a damage of pulmonary endothelial cells with subsequent oedema formation. Alterations in reactive oxygen species (ROS) have been suggested as a crucial mediator of such responses. Among the various sources of cellular ROS production, NADPH oxidases (NOXs) have been found to contribute to these physiological and pathophysiological signalling processes. NOXs are the only known examples that generate ROS as the primary function of the enzyme system. However, the downstream targets of NOX-derived ROS signalling in hypoxia are still not known. Canonical transient receptor potential (TRPC) channels recently have been recognised as directly or indirectly ROS-activated channels and have been shown to be essential for hypoxia-dependent vascular regulatory processes in the lung. Against this background, we here summarise the current knowledge on NOX-mediated TRPC channel signalling during hypoxia in the pulmonary circulation. PMID:26424109

  16. Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

    PubMed Central

    Williams, Robert; Lemaire, Paul; Lewis, Philip; McDonald, Fiona B.; Lucking, Eric; Hogan, Sean; Sheehan, David; Healy, Vincent; O'Halloran, Ken D.

    2015-01-01

    Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1? content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1? content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1?-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling. PMID:25688214

  17. Andrographolide inhibits TNF?-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells.

    PubMed

    Lu, Chia-Yang; Yang, Ya-Chen; Li, Chien-Chun; Liu, Kai-Li; Lii, Chong-Kuei; Chen, Haw-Wen

    2014-09-01

    Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNF?-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox), and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47(phox) abolished TNF?-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNF?-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent. PMID:24998495

  18. Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes.

    PubMed

    Gorin, Yves; Cavaglieri, Rita C; Khazim, Khaled; Lee, Doug-Yoon; Bruno, Francesca; Thakur, Sachin; Fanti, Paolo; Szyndralewiez, Cédric; Barnes, Jeffrey L; Block, Karen; Abboud, Hanna E

    2015-06-01

    Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes. PMID:25656366

  19. Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

    PubMed

    Chignalia, Andreia Z; Oliveira, Maria Aparecida; Debbas, Victor; Dull, Randal O; Laurindo, Francisco R M; Touyz, Rhian M; Carvalho, Maria Helena C; Fortes, Zuleica B; Tostes, Rita C

    2015-07-01

    The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk. PMID:25700020

  20. In vitro fructose exposure overactivates NADPH oxidase and causes oxidative stress in the isolated rat aorta.

    PubMed

    Almenara, Camila C P; Mill, José G; Vassallo, Dalton V; Baldo, Marcelo P; Padilha, Alessandra S

    2015-12-01

    Fructose acutely interferes with cardiovascular function in humans and in animals, but the mechanisms remain unclear. Thus, we tested whether fructose can affect endothelial function without the interference of its metabolic effect by exposing the rat aorta to a high fructose concentration and then evaluate the vascular responses to vasoactive agents. We observed that fructose exposure causes overactivation of NADPH oxidase, which enhances superoxide anion production and increases NO degradation. Additionally, the enhanced vasoconstrictor action of hydrogen peroxide might exacerbate contractile responses. This vasoactive imbalance might be the key role by which fructose induces vascular dysfunction. PMID:26320835

  1. Nox1 NADPH oxidase is necessary for late but not early myocardial ischaemic preconditioning

    PubMed Central

    Jiang, Shuxia; Streeter, Jennifer; Schickling, Brandon M.; Zimmerman, Kathy; Weiss, Robert M.; Miller, Francis J.

    2014-01-01

    Aims Ischaemic preconditioning (IPC) is an adaptive mechanism that renders the myocardium resistant to injury from subsequent hypoxia. Although reactive oxygen species (ROS) contribute to both the early and late phases of IPC, their enzymatic source and associated signalling events have not yet been understood completely. Our objective was to investigate the role of the Nox1 NADPH oxidase in cardioprotection provided by IPC. Methods and results Wild-type (WT) and Nox1-deficient mice were treated with three cycles of brief coronary occlusion and reperfusion, followed by prolonged occlusion either immediately (early IPC) or after 24 h (late IPC). Nox1 deficiency had no impact on the cardioprotection afforded by early IPC. In contrast, deficiency of Nox1 during late IPC resulted in a larger infarct size, cardiac remodelling, and increased myocardial apoptosis compared with WT hearts. Furthermore, expression of Nox1 in WT hearts increased in response to late IPC. Deficiency of Nox1 abrogated late IPC-mediated activation of cardiac nuclear factor-?B (NF-?B) and induction of tumour necrosis factor-? (TNF-?) in the heart and circulation. Finally, knockdown of Nox1 in cultured cardiomyocytes prevented TNF-? induction of NF-?B and the protective effect of IPC on hypoxia-induced apoptosis. Conclusions Our data identify a critical role for Nox1 in late IPC and define a previously unrecognized link between TNF-? and NF-?B in mediating tolerance to myocardial injury. These findings have clinical significance considering the emergence of Nox1 inhibitors for the treatment of cardiovascular disease. PMID:24501329

  2. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  3. 20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt

    PubMed Central

    Gruenloh, Stephanie K.; Gao, Ying; Manthati, Vijay L.; Dubasi, Narsimhaswamy; Falck, John R.; Medhora, Meetha; Jacobs, Elizabeth R.

    2010-01-01

    We have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide (H2O2) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Ser1179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on H2O2 as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs. PMID:20061439

  4. Renal denervation attenuates NADPH oxidase-mediated oxidative stress and hypertension in rats with hydronephrosis.

    PubMed

    Peleli, Maria; Al-Mashhadi, Ammar; Yang, Ting; Larsson, Erik; Wåhlin, Nils; Jensen, Boye L; G Persson, A Erik; Carlström, Mattias

    2016-01-01

    Hydronephrosis is associated with the development of salt-sensitive hypertension. Studies have suggested that increased sympathetic nerve activity and oxidative stress play important roles in hypertension and the modulation of salt sensitivity. The present study primarily aimed to examine the role of renal sympathetic nerve activity in the development of hypertension in rats with hydronephrosis. In addition, we aimed to investigate if NADPH oxidase (NOX) function could be affected by renal denervation. Partial unilateral ureteral obstruction (PUUO) was created in 3-wk-old rats to induce hydronephrosis. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high-, and low-salt diets. The renal excretion pattern, NOX activity, and expression as well as components of the renin-angiotensin-aldosterone system were characterized after treatment with the normal salt diet. On the normal salt diet, rats in the PUUO group had elevated blood pressure compared with control rats (115 ± 3 vs. 87 ± 1 mmHg, P < 0.05) and displayed increased urine production and lower urine osmolality. The blood pressure change in response to salt loading (salt sensitivity) was more pronounced in the PUUO group compared with the control group (15 ± 2 vs. 5 ± 1 mmHg, P < 0.05). Renal denervation in PUUO rats attenuated both hypertension (97 ± 3 mmHg) and salt sensitivity (5 ± 1 mmHg, P < 0.05) and normalized the renal excretion pattern, whereas the degree of renal fibrosis and inflammation was not changed. NOX activity and expression as well as renin and ANG II type 1A receptor expression were increased in the renal cortex from PUUO rats and normalized by denervation. Plasma Na(+) and K(+) levels were elevated in PUUO rats and normalized after renal denervation. Finally, denervation in PUUO rats was also associated with reduced NOX expression, superoxide production, and fibrosis in the heart. In conclusion, renal denervation attenuates hypertension and restores the renal excretion pattern, which is associated with reduced renal NOX and components of the renin-angiotensin-aldosterone system. This study emphasizes a link between renal nerves, the development of hypertension, and modulation of NOX function. PMID:26538440

  5. Role of NADPH oxidases and reactive oxygen species in regulation of bone turnover and the skeletal toxicity of alcohol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies with genetically modified mice and dietary antioxidants have suggested an important role for superoxide derived from NADPH oxidase (NOX) enzymes and other reactive oxygen species (ROS) such as hydrogen peroxide in regulation of normal bone turnover during development and also in the r...

  6. NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1?/G-CSF axis.

    PubMed

    Bagaitkar, Juhi; Pech, Nancy K; Ivanov, Stoyan; Austin, Anthony; Zeng, Melody Yue; Pallat, Sabine; Huang, Guangming; Randolph, Gwendalyn J; Dinauer, Mary C

    2015-12-17

    The leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates reactive oxygen species essential in microbial killing and regulation of inflammation. Inactivating mutations in this enzyme lead to chronic granulomatous disease (CGD), associated with increased susceptibility to both pyogenic infections and to inflammatory disorders. The role of the NADPH oxidase in regulating inflammation driven by nonmicrobial stimuli is poorly understood. Here, we show that NADPH oxidase deficiency enhances the early local release of interleukin-1? (IL-1?) in response to damaged cells, promoting an excessive granulocyte colony-stimulating factor (G-CSF)-regulated neutrophilic response and prolonged inflammation. In peritoneal inflammation elicited by tissue injury, X-linked Cybb-null (X-CGD) mice exhibited increased release of IL-1? and IL-1 receptor -mediated G-CSF production. In turn, higher levels of systemic G-CSF increased peripheral neutrophilia, which amplified neutrophilic peritoneal inflammation in X-CGD mice. Dampening early neutrophil recruitment by neutralization of IL-1?, G-CSF, or neutrophil depletion itself promoted resolution of otherwise prolonged inflammation in X-CGD. IL-1? played little role. Thus, we identified an excessive IL-1?/G-CSF response as a major driver of enhanced sterile inflammation in CGD in the response to damaged cells. More broadly, these results provide new insights into the regulation of sterile inflammation, and identify the NADPH oxidase in regulating the amplitude of the early neutrophilic response. PMID:26443623

  7. Fibrillar beta-amyloid peptide Abeta1-40activates microglial proliferation via stimulating TNF-alpha release and H2O2derived from NADPH oxidase: a cell culture study

    E-print Network

    Jekabsone, Aiste; Mander, Palwinder K.; Tickler, Anna; Sharpe, Martyn; Brown, Guy C.

    2006-09-07

    1997, 56:321-339. 3. Zekry D, Epperson TK, Krause KH: A role for NOX NADPH oxi- dases in Alzheimer's disease and other types of dementia? IUBMB Life 2003, 55:307-313. 4. McGeer EG, Yasojima K, Schwab C, McGeer PL: The pentraxins: possible role... proliferation of human vascular endothelial cells. J Am Soc Nephrol 2000, 11:1819-1825. 30. Fialkow L, Chan CK, Downey GP: Inhibition of CD45 during neu- trophil activation. J Immunol 1997, 158:5409-5417. 31. Suh HS, Kim MO, Lee SC: Inhibition of granulocyte...

  8. Protein disulfide isomerase expression increases in resistance arteries during hypertension development. Effects on Nox1 NADPH oxidase signaling

    PubMed Central

    Androwiki, Aline C. D.; Camargo, Lívia de Lucca; Sartoretto, Simone; Couto, Gisele K.; Ribeiro, Izabela M. R.; Veríssimo-Filho, Sidney; Rossoni, Luciana V.; Lopes, Lucia R.

    2015-01-01

    NADPH oxidases derived reactive oxygen species (ROS) play an important role in vascular function and remodeling in hypertension through redox signaling processes. Previous studies demonstrated that protein disulfide isomerase (PDI) regulates Nox1 expression and ROS generation in cultured vascular smooth muscle cells. However, the role of PDI in conductance and resistance arteries during hypertension development remains unknown. The aim of the present study was to investigate PDI expression and NADPH oxidase dependent ROS generation during hypertension development. Mesenteric resistance arteries (MRA) and thoracic aorta were isolated from 6, 8, and 12 week-old spontaneously hypertensive (SHR) and Wistar rats. ROS production (dihydroethidium fluorescence), PDI (WB, imunofluorescence), Nox1 and NOX4 (RT-PCR) expression were evaluated. Results show a progressive increase in ROS generation in MRA and aorta from 8 to 12 week-old SHR. This effect was associated with a concomitant increase in PDI and Nox1 expression only in MRA. Therefore, suggesting a positive correlation between PDI and Nox1 expression during the development of hypertension in MRA. In order to investigate if this effect was due to an increase in arterial blood pressure, pre hypertensive SHR were treated with losartan (20 mg/kg/day for 30 days), an AT1 receptor antagonist. Losartan decreased blood pressure and ROS generation in both vascular beds. However, only in SHR MRA losartan treatment lowered PDI and Nox1 expression to control levels. In MRA PDI inhibition (bacitracin, 0.5 mM) decreased Ang II redox signaling (p-ERK 1/2). Altogether, our results suggest that PDI plays a role in triggering oxidative stress and vascular dysfunction in resistance but not in conductance arteries, increasing Nox1 expression and activity. Therefore, PDI could be a new player in oxidative stress and functional alterations in resistance arteries during the establishment of hypertension. PMID:25870854

  9. Crystallization and preliminary crystallographic analysis of p40{sup phox}, a regulatory subunit of NADPH oxidase

    SciTech Connect

    Honbou, Kazuya; Yuzawa, Satoru; Suzuki, Nobuo N.; Fujioka, Yuko; Sumimoto, Hideki; Inagaki, Fuyuhiko

    2006-10-01

    Human p40{sup phox} was expressed, purified and crystallized. Diffraction data were collected to a resolution of 3.0 Å. p40{sup phox} is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for production of the superoxide that kills invasive microorganisms. Full-length p40{sup phox} was expressed in Escherichia coli, purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 3.0 Å resolution at 100 K using synchrotron radiation. The crystal belongs to space group C222{sub 1}, with unit-cell parameters a = 146.27, b = 189.81, c = 79.88 Å. This crystal was estimated to contain two or three protein molecules per asymmetric unit from the acceptable range of volume-to-weight ratio values.

  10. Hypercholesterolemia-induced erectile dysfunction: endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase

    PubMed Central

    Musicki, Biljana; Liu, Tongyun; Lagoda, Gwen A.; Strong, Travis D.; Sezen, Sena F.; Johnson, Justin M.; Burnett, Arthur L.

    2010-01-01

    INTRODUCTION Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood. AIMS We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED. METHODS Low-density-lipoprotein receptor (LDLR)–null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67phox, p47phox, and gp91phox), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes. MAIN OUTCOME MEASURES Molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED. RESULTS Erectile response was significantly (P<0.05) reduced in hypercholesterolemic LDLR-null mice compared to WT mice. Relative to WT mice, hypercholesterolemia increased (P<0.05) protein expressions of NAD(P)H oxidase subunits p67phox, p47phox and gp91phox, eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P<0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P<0.05) maximal intracavernosal pressure, and reversed (P < 0.05) the abnormalities in protein expressions of gp67phox and gp47phox, 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia. CONCLUSION Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED. PMID:20626609

  11. Which NADPH Oxidase Isoform Is Relevant for Ischemic Stroke? The Case for Nox 2

    PubMed Central

    Kahles, Timo

    2013-01-01

    Abstract Significance and Recent Advances: Ischemic stroke is the leading cause of disability and third in mortality in industrialized nations. Immediate restoration of cerebral blood flow is crucial to salvage brain tissue, but only few patients are eligible for recanalization therapy. Thus, the need for alternative neuroprotective strategies is huge, and antioxidant interventions have long been studied in this context. Reactive oxygen species (ROS) physiologically serve as signaling molecules, but excessive amounts of ROS, as generated during ischemia/reperfusion (I/R), contribute to tissue injury. Critical Issues: Nevertheless and despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or do not reach the crucial target structures of ROS-induced injury effectively. Future Directions: Thus, a potentially more promising approach is the inhibition of the source of disease-promoting ROS. Within recent years, NADPH oxidases (Nox) of the Nox family have been identified as mediators of neuronal pathology. As, however, several Nox homologs are expressed in neuronal tissue, and as many of the pharmacological inhibitors employed are rather unspecific, the concept of Nox as mediators of brain damage is far from being settled. In this review, we will discuss the contribution of Nox homologs to I/R injury at large as well as to neuronal damage in particular. We will illustrate that the current data provide evidence for Nox2 as the most important NADPH oxidase mediating cerebral injury. Antioxid. Redox Signal. 18, 1400–1417. PMID:22746273

  12. Fluid shear stress upregulates placental growth factor in the vessel wall via NADPH oxidase 4.

    PubMed

    Rashdan, Nabil A; Lloyd, Pamela G

    2015-11-15

    Placental growth factor (PLGF), a potent stimulator of arteriogenesis, is upregulated during outward arterial remodeling. Increased fluid shear stress (FSS) is a key physiological stimulus for arteriogenesis. However, the role of FSS in regulating PLGF expression is unknown. To test the hypothesis that FSS regulates PLGF expression in vascular cells and to identify the signaling pathways involved, human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells were cultured on either side of porous Transwell inserts. HCAEC were then exposed to pulsatile FSS of 0.07 Pa ("normal," mimicking flow through quiescent collaterals), 1.24 Pa ("high," mimicking increased flow in remodeling collaterals), or 0.00 Pa ("static") for 2 h. High FSS increased secreted PLGF protein ?1.4-fold compared with static control (n = 5, P < 0.01), while normal FSS had no significant effect on PLGF. Similarly, high flow stimulated PLGF mRNA expression nearly twofold in isolated mouse mesenteric arterioles. PLGF knockdown using siRNA revealed that HCAEC were the primary source of PLGF in cocultures (n = 5, P < 0.01). Both H2O2 and nitric oxide production were increased by FSS compared with static control (n = 5, P < 0.05). N(G)-nitro-l-arginine methyl ester (100 ?M) had no significant effect on the FSS-induced increase in PLGF. In contrast, both catalase (500 U/ml) and diphenyleneiodonium (5 ?M) attenuated the effects of FSS on PLGF protein in cocultures. Diphenyleneiodonium also blocked the effect of high flow to upregulate PLGF mRNA in isolated arterioles. Further studies identified NADPH oxidase 4 as a source of reactive oxygen species for this pathway. We conclude that FSS regulates PLGF expression via NADPH oxidase 4 and reactive oxygen species signaling. PMID:26408539

  13. Role of NADPH oxidase in interleukin-4-induced monocyte chemoattractant protein-1 expression in vascular endothelium

    PubMed Central

    Lee, Won Hee; Kim, Paul H.

    2010-01-01

    Objective and design The pro-oxidative and pro-inflammatory pathways in vascular endothelium have been implicated in the development of atherosclerosis. In the present study, we investigated effect of interleukin-4 (IL-4) on monocyte chemoattractant protein-1 (MCP-1) expression in vascular endothelium and examined the role of distinct sources of reactive oxygen species (ROS) in this process. Methods and results Real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that IL-4 significantly up-regulated mRNA and protein expression of MCP-1 in human aortic endothelial cells (HAEC) and C57BL/6 mice. A significant and dose-dependent inhibition of IL-4-induced MCP-1 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate and epigallocatechin gallate, indicating that IL-4-induced MCP-1 expression is mediated via a ROS-dependent mechanism. Additionally, pharmacological inhibitors of NADPH oxidase (NOX) significantly attenuated IL-4-induced MCP-1 expression in HAEC. Furthermore, the disruption of the NOX gene dramatically reduced IL-4-induced MCP-1 expression in NOX knockout mice (B6.129S6-Cybbtm1Din/J). In contrast, overexpression of MCP-1 in IL-4-stimulated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase and the mitochondrial electron transport chain. Conclusions These results demonstrate that IL-4 up-regulates MCP-1 expression in vascular endothelium through NOX-mediated ROS generation. PMID:20349326

  14. LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor

    PubMed Central

    Zhu, Yinghui; Sun, Rui; Fang, Yujing; Fan, Yuhua; Xu, Fei

    2015-01-01

    Oxidative stress is thought to contribute to cancer development. Epstein–Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt’s lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy. PMID:26244812

  15. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    SciTech Connect

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-12-19

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91{sup phox} was dose-dependent. Meanwhile, the cytoplasmic subunit p47{sup phox} was translocated to the cell membrane and localized with p22{sup phox} and gp91{sup phox} to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  16. Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes.

    PubMed

    Park, Youngjin; Stanley, David W; Kim, Yonggyun

    2015-08-01

    Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4. PMID:26071791

  17. Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

    PubMed

    Maeda, Morihiko; Hayashi, Toshio; Mizuno, Natsumi; Hattori, Yuichi; Kuzuya, Masafumi

    2015-01-01

    Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-?-galactosidase (SA-?-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-?-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction. PMID:25879533

  18. Anandamide Protects HT22 Cells Exposed to Hydrogen Peroxide by Inhibiting CB1 Receptor-Mediated Type 2 NADPH Oxidase

    PubMed Central

    Jia, Ji; Wu, Mingchun; Zhang, Lei; Zhang, Xiajing; Zhai, Qian; Jiang, Tao; Xiong, Lize

    2014-01-01

    Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells. PMID:25136404

  19. Intermittent High Glucose Implements Stress-Induced Senescence in Human Vascular Endothelial Cells: Role of Superoxide Production by NADPH Oxidase

    PubMed Central

    Maeda, Morihiko; Hayashi, Toshio; Mizuno, Natsumi; Hattori, Yuichi; Kuzuya, Masafumi

    2015-01-01

    Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-?-galactosidase(SA-?-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-?-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction. PMID:25879533

  20. Amyloid ?25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

    PubMed

    Part, Kristin; Künnis-Beres, Kai; Poska, Helen; Land, Tiit; Shimmo, Ruth; Zetterström Fernaeus, Sandra

    2015-12-10

    Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (A?) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of A? through ROS and metals are mainly studied in neuronal cells using full-length A?1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain A?25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if A?25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that A?25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained A?25-35 induced ROS. PMID:26505916

  1. Histamine Promotes the Development of Monocyte-Derived Dendritic Cells and Reduces Tumor Growth by Targeting the Myeloid NADPH Oxidase

    PubMed Central

    Wiktorin, Hanna G.; Lenox, Brianna; Ewald Sander, Frida; Aydin, Ebru; Aurelius, Johan; Thorén, Fredrik B.; Ståhlberg, Anders; Hermodsson, Svante; Hellstrand, Kristoffer

    2015-01-01

    The efficiency of immune-mediated clearance of cancer cells is hampered by immunosuppressive mediators in the malignant microenvironment, including NADPH oxidase–derived reactive oxygen species. We aimed at defining the effects of histamine, an inhibitor of the myeloid NADPH oxidase/NOX2, on the development of Ag-presenting dendritic cells (DCs) from myeloid precursors and the impact of these mechanisms for tumor growth. Histamine was found to promote the maturation of human DCs from monocytes by increasing the expression of HLA-DR and costimulatory molecules, which resulted in improved induction of Th cells with Th0 polarity. Experiments using wild-type and NOX2-deficient myelomonoblastic cells showed that histamine facilitated myeloid cell maturation only in cells capable of generating reactive oxygen species. Treatment of mice with histamine reduced the growth of murine EL-4 lymphomas in parallel with an increment of tumor-infiltrating DCs in NOX2-sufficient mice but not in NOX2-deficient (gp91phox?/?) mice. We propose that strategies to target the myeloid NADPH oxidase may facilitate the development of endogenous DCs in cancer. PMID:25870245

  2. EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: evidence for NADPH oxidase and mitochondrial stimulation.

    PubMed

    Culcasi, Marcel; Benameur, Laila; Mercier, Anne; Lucchesi, Céline; Rahmouni, Hidayat; Asteian, Alice; Casano, Gilles; Botta, Alain; Kovacic, Hervé; Pietri, Sylvia

    2012-09-30

    To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24 h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo. PMID:22940227

  3. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  4. Neonatal hyperglycemia induces oxidative stress in the rat brain: the role of pentose phosphate pathway enzymes and NADPH oxidase.

    PubMed

    Rosa, Andrea Pereira; Jacques, Carlos Eduardo Dias; de Souza, Laila Oliveira; Bitencourt, Fernanda; Mazzola, Priscila Nicolao; Coelho, Juliana Gonzales; Mescka, Caroline Paula; Dutra-Filho, Carlos Severo

    2015-05-01

    Recently, the consequences of diabetes on the central nervous system (CNS) have received great attention. However, the mechanisms by which hyperglycemia affects the central nervous system remain poorly understood. In addition, recent studies have shown that hyperglycemia induces oxidative damage in the adult rat brain. In this regard, no study has assessed oxidative stress as a possible mechanism that affects the brain normal function in neonatal hyperglycemic rats. Thus, the present study aimed to investigate whether neonatal hyperglycemia elicits oxidative stress in the brain of neonate rats subjected to a streptozotocin-induced neonatal hyperglycemia model (5-day-old rats). The activities of glucose-6-phosphate-dehydrogenase (G6PD), 6-phosphogluconate-dehydrogenase (6-PGD), NADPH oxidase (Nox), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), the production of superoxide anion, the thiobarbituric acid-reactive substances (TBA-RS), and the protein carbonyl content were measured. Neonatal hyperglycemic rats presented increased activities of G6PD, 6PGD, and Nox, which altogether may be responsible for the enhanced production of superoxide radical anion that was observed. The enhanced antioxidant enzyme activities (SOD, CAT, and GSHPx) that were observed in neonatal hyperglycemic rats, which may be caused by a rebound effect of oxidative stress, were not able to hinder the observed lipid peroxidation (TBA-RS) and protein damage in the brain. Consequently, these results suggest that oxidative stress could represent a mechanism that explains the harmful effects of neonatal hyperglycemia on the CNS. PMID:25682169

  5. The Role of the NADPH Oxidase NOX2 in Prion Pathogenesis

    PubMed Central

    Sorce, Silvia; Nuvolone, Mario; Keller, Annika; Falsig, Jeppe; Varol, Ahmet; Schwarz, Petra; Bieri, Monika; Budka, Herbert; Aguzzi, Adriano

    2014-01-01

    Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis. PMID:25502554

  6. NADPH oxidases 1 and 4 mediate cellular senescence induced by resveratrol in human endothelial cells.

    PubMed

    Schilder, Yvonne D C; Heiss, Elke H; Schachner, Daniel; Ziegler, Jürgen; Reznicek, Gottfried; Sorescu, Dan; Dirsch, Verena M

    2009-06-15

    Resveratrol is believed to be partially responsible for the French paradox--the low risk of cardiovascular disease despite a high-fat diet in the French population. Recently, resveratrol has also been discussed as a life-span booster in several organisms. Age-related diseases are associated on the cellular level with senescence. We, therefore, hypothesized that resveratrol is vasoprotective by counteracting endothelial cell senescence. Surprisingly, we observed that chronic treatment with resveratrol (10 microM) was prosenescent in primary human endothelial cells. Resveratrol induced elevated reactive oxygen species (ROS) levels that were associated with and causally linked to an accumulation of cells in the S phase of the cell cycle, as measured by flow cytometry. We further show that cell accumulation in S phase leads to increased ROS and finally senescence. Using an siRNA approach, we clearly identified two NADPH oxidases, Nox1 and Nox4, as major targets of resveratrol and primary sources of ROS that act upstream of the observed S-phase accumulation. PMID:19328228

  7. Role of Neuronal NADPH Oxidase 1 in the Peri-Infarct Regions after Stroke

    PubMed Central

    Choi, Dong-Hee; Kim, Ji-Hye; Lee, Kyoung-Hee; Kim, Hahn-Young; Kim, Yoon-Seong; Choi, Wahn Soo; Lee, Jongmin

    2015-01-01

    The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia—reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2?-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke. PMID:25617620

  8. NADPH Oxidase Enzymes in Skin Fibrosis: Molecular Targets and Therapeutic Agents

    PubMed Central

    Lev-Tov, Hadar; Jagdeo, Jared

    2013-01-01

    Fibrosis is characterized by the excessive deposition of extracellular matrix components eventually resulting in organ dysfunction and failure. In dermatology, fibrosis is the hallmark component of many skin diseases, including systemic sclerosis, graft versus host disease, hypertrophic scars, keloids, nephrogenic systemic fibrosis, porphyria cutanea tarda, restrictive dermopathy and other conditions. Fibrotic skin disorders may be debilitating and impair quality of life. There are few FDA-approved anti-fibrotic drugs; thus, research in this area is crucial in addressing this deficiency. Recent investigations elucidating the pathogenesis of skin fibrosis have implicated endogenous reactive oxygen species produced by the multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzyme complex. In this review we discuss Nox enzymes and their role in skin fibrosis. An overview of the Nox enzyme family is presented and their role in the pathogenesis of skin fibrosis is discussed. The mechanisms that Nox enzymes influence specific skin fibrotic disorders are also reviewed. Finally, we describe the therapeutic approaches to ameliorate skin fibrosis by directly targeting Nox enzymes with the use of statins, p47phox subunit modulators, or GKT137831, a competitive inhibitor of Nox enzymes. Nox enzymes can also be targeted indirectly via scavenging ROS with antioxidants. We believe that Nox modulators are worthy of further investigation and have the potential to transform the management of skin fibrosis by dermatologists. PMID:24155025

  9. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase.

    PubMed

    Sorescu, George P; Song, Hannah; Tressel, Sarah L; Hwang, Jinah; Dikalov, Sergey; Smith, Debra A; Boyd, Nolan L; Platt, Manu O; Lassègue, Bernard; Griendling, Kathy K; Jo, Hanjoong

    2004-10-15

    Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. PMID:15388638

  10. Oxidative stress, redox signalling and endothelial dysfunction in ageing-related neurodegenerative diseases: a role of NADPH oxidase 2

    PubMed Central

    Cahill-Smith, Sarah; Li, Jian-Mei

    2014-01-01

    Chronic oxidative stress and oxidative damage of the cerebral microvasculature and brain cells has become one of the most convincing theories in neurodegenerative pathology. Controlled oxidative metabolism and redox signalling in the central nervous system are crucial for maintaining brain function; however, excessive production of reactive oxygen species and enhanced redox signalling damage neurons. While several enzymes and metabolic processes can generate intracellular reactive oxygen species in the brain, recently an O2?-generating enzyme, NADPH oxidase 2 (Nox2), has emerged as a major source of oxidative stress in ageing-related vascular endothelial dysfunction and neurodegenerative diseases. The currently available inhibitors of Nox2 are not specific, and general antioxidant therapy is not effective in the clinic; therefore, insights into the mechanism of Nox2 activation and its signalling pathways are needed for the discovery of novel drug targets to prevent or treat these neurodegenerative diseases. This review summarizes the recent developments in understanding the mechanisms of Nox2 activation and redox-sensitive signalling pathways and biomarkers involved in the pathophysiology of the most common neurodegenerative diseases, such as ageing-related mild cognitive impairment, Alzheimer’s disease and Parkinson’s disease. PMID:25279404

  11. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model

    PubMed Central

    Harraz, Maged M.; Marden, Jennifer J.; Zhou, Weihong; Zhang, Yulong; Williams, Aislinn; Sharov, Victor S.; Nelson, Kathryn; Luo, Meihui; Paulson, Henry; Schöneich, Christian; Engelhardt, John F.

    2008-01-01

    Neurodegeneration in familial amyotrophic lateral sclerosis (ALS) is associated with enhanced redox stress caused by dominant mutations in superoxide dismutase–1 (SOD1). SOD1 is a cytosolic enzyme that facilitates the conversion of superoxide (O2•–) to H2O2. Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase–dependent (Nox-dependent) O2•– production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H2O2 uncoupled SOD1 binding in a reversible fashion, producing a self-regulating redox sensor for Nox-derived O2•– production. This process of redox-sensitive uncoupling of SOD1 from Rac1 was defective in SOD1 ALS mutants, leading to enhanced Rac1/Nox activation in transgenic mouse tissues and cell lines expressing ALS SOD1 mutants. Glial cell toxicity associated with expression of SOD1 mutants in culture was significantly attenuated by treatment with the Nox inhibitor apocynin. Treatment of ALS mice with apocynin also significantly increased their average life span. This redox sensor mechanism may explain the gain-of-function seen with certain SOD1 mutations associated with ALS and defines new therapeutic targets. PMID:18219391

  12. Effect of staurosporine in the morphology and viability of cerebellar astrocytes: role of reactive oxygen species and NADPH oxidase.

    PubMed

    Olguín-Albuerne, Mauricio; Domínguez, Guadalupe; Morán, Julio

    2014-01-01

    Cell death implies morphological changes that may contribute to the progression of this process. In astrocytes, the mechanisms involving the cytoskeletal changes during cell death are not well explored. Although NADPH oxidase (NOX) has been described as being a critical factor in the production of ROS, not much information is available about the participation of NOX-derived ROS in the cell death of astrocytes and their role in the alterations of the cytoskeleton during the death of astrocytes. In this study, we have evaluated the participation of ROS in the death of cultured cerebellar astrocytes using staurosporine (St) as death inductor. We found that astrocytes express NOX1, NOX2, and NOX4. Also, St induced an early ROS production and NOX activation that participate in the death of astrocytes. These findings suggest that ROS produced by St is generated through NOX1 and NOX4. Finally, we showed that the reorganization of tubulin and actin induced by St is ROS independent and that St did not change the level of expression of these cytoskeletal proteins. We conclude that ROS produced by a NOX is required for cell death in astrocytes, but not for the morphological alterations induced by St. PMID:25215174

  13. NADPH Oxidase-Mediated Superoxide Production by Intermediary Bacterial Metabolites of Dibenzofuran: A Potential Cause for Trans-Mitochondrial Membrane Potential (??m) Collapse in Human Hepatoma Cells.

    PubMed

    Jaiswal, Prashant Kumar; Gupta, Jyotsana; Shahni, Shweta; Thakur, Indu Shekhar

    2015-09-01

    Dibenzofuran is a direct precursor of extremely toxic compounds such as dioxins. It is widely distributed persistent organic pollutant in environment that potentiate oxidative stress, apoptosis, and necrosis through bioactivation in HepG2 cells. An alkalotolerent Pseudomonas strain ISTDF1 can metabolize dibenzofuran as a sole source of carbon and energy through diverse dioxygenation. However, there is a paucity of information about the potential toxic effects of the intermediary metabolites that are formed during treatment with dibenzofuran. We have assessed and discovered the potential mechanism of toxicity induced by metabolites of dibenzofuran that were formed at 18 and 36?h. Cell viability, CYP1A2 induction, ROS activity, Superoxide production, mitochondrial NADPH oxidase activity, and mitochondrial trans-membrane potential were studied using different assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), confocal laser scanning microscopy, and flow cytometry. Analysis revealed formation of 2-(1-carbonyl methylidine)-2,3-dihydrobenzofuranlidene after 18?h of bacterial treatment due to oxygenation at carbon (C3-C4). This compound induces higher mitochondrial NADPH oxidase-dependent superoxide production that makes it more toxic than the parent compound. It was evident that after 36?h of bacterial treatment, toxicity induced by dibenzofuran and its metabolites was completely removed. This study highlights the fact that despite of efficient biodegradation of toxicants, bioactive toxic intermediates can be formed. Therefore, it is necessary to assess the toxicity of each intermediary for complete mitigation of associated risk. PMID:26032510

  14. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells.

    PubMed

    Stanicka, Joanna; Russell, Eileen G; Woolley, John F; Cotter, Thomas G

    2015-04-10

    Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability. PMID:25697362

  15. NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

    PubMed Central

    Gonzalez, Daniel R.; Treuer, Adriana V.; Lamirault, Guillaume; Mayo, Vera; Cao, Yenong; Dulce, Raul A.

    2014-01-01

    Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca2+ concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca2+ content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy. PMID:25015966

  16. Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

    PubMed

    Ben Rejeb, Kilani; Lefebvre-De Vos, Delphine; Le Disquet, Isabel; Leprince, Anne-Sophie; Bordenave, Marianne; Maldiney, Régis; Jdey, Asma; Abdelly, Chedly; Savouré, Arnould

    2015-12-01

    Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of ?(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant. PMID:26180024

  17. beta-aminobutyric acid primes an NADPH oxidase-dependent reactive oxygen species production during grapevine-triggered immunity.

    PubMed

    Dubreuil-Maurizi, Carole; Trouvelot, Sophie; Frettinger, Patrick; Pugin, Alain; Wendehenne, David; Poinssot, Benoît

    2010-08-01

    The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase-dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem. PMID:20615112

  18. NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts.

    PubMed

    Cucoranu, Ioan; Clempus, Roza; Dikalova, Anna; Phelan, Patrick J; Ariyan, Srividya; Dikalov, Sergey; Sorescu, Dan

    2005-10-28

    Human cardiac fibroblasts are the main source of cardiac fibrosis associated with cardiac hypertrophy and heart failure. Transforming growth factor-beta1 (TGF-beta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (SM alpha-actin) de novo and produce extracellular matrix. We hypothesized that TGF-beta1-stimulated conversion of fibroblasts to myofibroblasts requires reactive oxygen species derived from NAD(P)H oxidases (Nox). We found that TGF-beta1 potently upregulates the contractile marker SM alpha-actin mRNA (7.5+/-0.8-fold versus control). To determine whether Nox enzymes are involved, we first performed quantitative real time polymerase chain reaction and found that Nox5 and Nox4 are abundantly expressed in cardiac fibroblasts, whereas Nox1 and Nox2 are barely detectable. On stimulation with TGF-beta1, Nox4 mRNA is dramatically upregulated by 16.2+/-0.8-fold (n=3, P<0.005), whereas Nox5 is downregulated. Small interference RNA against Nox4 downregulates Nox4 mRNA by 80+/-5%, inhibits NADPH-driven superoxide production in response to TGF-beta1 by 65+/-7%, and reduces TGF-beta1-induced expression of SM alpha-actin by 95+/-2% (n=6, P<0.05). Because activation of small mothers against decapentaplegic (Smads) 2/3 is critical for myofibroblast conversion in response to TGF-beta1, we also determined whether Nox4 affects Smad 2/3 phosphorylation. Depletion of Nox4 but not Nox5 inhibits baseline and TGF-beta1 stimulation of Smad 2/3 phosphorylation by 75+/-5% and 68+/-3%, respectively (n=7, P<0.0001). We conclude that Nox 4 mediates TGF-beta1-induced conversion of fibroblasts to myofibroblasts by regulating Smad 2/3 activation. Thus, Nox4 may play a critical role in the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with human heart failure. PMID:16179589

  19. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    SciTech Connect

    Eum, Sung Yong Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-10-15

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  20. Hypoxia-Inducible Factor 1 Mediates Increased Expression of NADPH Oxidase-2 in Response to Intermittent Hypoxia

    PubMed Central

    Yuan, Guoxiang; Khan, Shakil A.; Luo, Weibo; Nanduri, Jayasri; Semenza, Gregg L.; Prabhakar, Nanduri R.

    2015-01-01

    Sleep-disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro-oxidant enzymes, especially NADPH oxidase-2 (Nox2). Previous studies showed that (i) IH activates hypoxia-inducible factor 1 (HIF-1) in a ROS-dependent manner and (ii) HIF-1 is required for IH-induced ROS generation, indicating the existence of a feed-forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH-induced expression of Nox2 is mediated by HIF-1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild-type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF-1 activity through RNA interference or pharmacologic inhibition (digoxin or YC-1) or by genetic knockout of HIF-1? in MEFs. Increasing HIF-1? expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF-1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild-type mice to IH (8 h/day for 10 days) up-regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a+/? mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF-1, ROS generation, and cardiovascular pathology in response to IH. PMID:21302291

  1. Involvement of NADPH oxidase and iNOS in rodent pulmonary cytokine responses to urban air and mineral particles.

    PubMed

    Becher, Rune; Bucht, Anders; Øvrevik, Johan; Hongslo, Jan K; Dahlman, Hans Jørgen; Samuelsen, Jan Tore; Schwarze, Per E

    2007-06-01

    We have investigated the potential of two complex mineral particles (feldspar and mylonite), quartz (Min-U-Sil), and suspended particulate matter (SRM-1648) (SPM) from urban air to induce inflammatory cytokine responses in primary rat alveolar type 2 cells and alveolar macrophages, and the involvement of cellular formation of free radicals in these responses. All particle types induced an increased release of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2 from type 2 cells. Diphenyleneiodonium chloride (DPI), a selective inhibitor of NADPH-oxidase, reduced the IL-6 and MIP-2 responses to quartz, SPM and mylonite. N-(3-[Aminomethyl] benzyl) acetamidine (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), significantly reduced the Il-6 response to SPM and feldspar in the type 2 cells. The macrophages displayed significantly increased TNF-alpha and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-alpha and MIP-2 responses to quartz, and the MIP-2 response to SPM. No significant effect of 1400 W was detected in the alveolar macrophages. The role of particle-induced cellular generation of free radicals in lung cytokine responses was further elucidated in mice that lacked either NADPH-oxidase or iNOS as well as in wild-type (wt) mice. All particles were able to elicit increased cytokine levels in the bronchoalveolar lavage (BAL) fluid of the mice, although the levels depended on particle type. The NADPH-oxidase knockout (KO) mice demonstrated a significantly lower IL-6 and MIP-2 responses to SPM compared to their respective wt mice. The iNOS KO mice displayed significantly reduced IL-6, TNF-alpha, and MIP-2 responses to SPM. The overall results indicate the involvement of cellular free-radical formation in the pulmonary cytokine responses to particles of varying composition. PMID:17510837

  2. Acute restraint stress increases carotid reactivity in type-I diabetic rats by enhancing Nox4/NADPH oxidase functionality.

    PubMed

    Moreira, Josimar D; Pernomian, Larissa; Gomes, Mayara S; Pernomian, Laena; Moreira, Rafael P; do Prado, Alejandro F; da Silva, Carlos H T P; de Oliveira, Ana M

    2015-10-15

    Hyperglycemia increases the generation of reactive oxygen species and affects systems that regulate the vascular tone including renin-angiotensin system. Stress could exacerbate intracellular oxidative stress during Diabetes upon the activation of angiotensin AT1/NADPH oxidase pathway, which contributes to the development of diabetic cardiovascular complications. For this study, type-I Diabetes was induced in Wistar rats by intraperitoneal injection of streptozotocin. 28 days after streptozotocin injection, the animals underwent to acute restraint stress for 3h. Cumulative concentration-response curves for angiotensin II were obtained in carotid rings pre-treated or not with Nox or cyclooxygenase inhibitors. Nox1 or Nox4 expression and activity were assessed by Western blotting and lucigenin chemiluminescence, respectively. The role of Nox1 and Nox4 on reactive oxygen species generation was evaluated by flow cytometry and Amplex Red assays. Cyclooxygenases expression was assessed by real-time polymerase chain reaction. The contractile response evoked by angiotensin II was increased in diabetic rat carotid. Acute restraint stress increased this response in this vessel by mechanisms mediated by Nox4, whose local expression and activity in generating hydrogen peroxide are increased. The contractile hyperreactivity to angiotensin II in stressed diabetic rat carotid is also mediated by metabolites derived from cyclooxygenase-2, whose local expression is increased. Taken together, our findings suggest that acute restraint stress exacerbates the contractile hyperreactivity to angiotensin II in diabetic rat carotid by enhancing Nox4-driven generation of hydrogen peroxide, which evokes contractile tone by cyclooxygenases-dependent mechanisms. Finally, these findings highlight the harmful role played by acute stress in modulating diabetic vascular complications. PMID:26387612

  3. A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis.

    PubMed

    Jiang, Lulu; Chen, Shih-Heng; Chu, Chun-Hsien; Wang, Shi-Jun; Oyarzabal, Esteban; Wilson, Belinda; Sanders, Virginia; Xie, Keqin; Wang, Qingshan; Hong, Jau-Shyong

    2015-06-01

    Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. It was found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from ?2-adrenergic receptor (?2-AR)-deficient mice, suggesting that novel pathways other than ?2-AR are involved. To this end, It was found that submicromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (-) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism. PMID:25740080

  4. Molecular Mechanisms of the Crosstalk Between Mitochondria and NADPH Oxidase Through Reactive Oxygen Species—Studies in White Blood Cells and in Animal Models

    PubMed Central

    Kröller-Schön, Swenja; Steven, Sebastian; Kossmann, Sabine; Scholz, Alexander; Daub, Steffen; Oelze, Matthias; Xia, Ning; Hausding, Michael; Mikhed, Yuliya; Zinßius, Elena; Mader, Michael; Stamm, Paul; Treiber, Nicolai; Scharffetter-Kochanek, Karin; Li, Huige; Schulz, Eberhard; Wenzel, Philip; Münzel, Thomas

    2014-01-01

    Abstract Aims: Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. Results: NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD+/?) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD+/? mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. Innovation: These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. Conclusions: Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension. Antioxid. Redox Signal. 20, 247–266. PMID:23845067

  5. Intermittent Hypoxia-Induced Parvalbumin-Immunoreactive Interneurons Loss and Neurobehavioral Impairment is Mediated by NADPH-Oxidase-2.

    PubMed

    Yuan, Liang; Wu, Jing; Liu, Jiang; Li, Guowei; Liang, Dong

    2015-06-01

    Obstructive sleep apnea usually contribute to psychiatric diseases and cognitive impairments in adults. Loss of parvalbumin (PV)-immunoreactive interneurons (PV-IN) in the brain cortex is an important feature of psychiatric diseases, such as schizophrenia. Here we investigate the causal contribution of oxidative stress in the brain cortex to neuropathological alterations in a mouse model of sleep apnea. Wild-type (WT) and the NADPH-oxidase-2 (gp91-phox/NOX2) knock-out adult male C57BL/6J mice were exposed to intermittent hypoxia (IH) or standard room air in the same chamber. In vivo we determined the impact (1) of IH exposures on NOX2 expression, (2) of genetic gp91-phox/NOX2 knock-out and (3) of pharmacological NOX2 inhibition on IH-induced neuropathological alterations in adult mice. Endpoints were oxidative stress, PV-IN and neurobehavioral alterations. The results showed IH exposures increased NOX2 expression in the prefrontal cortex of WT mice, which was accompanied with elevations of indirect markers of oxidative stress (HNE, HIF-1?, 8-OHDG). WT mice showed loss of PV-IN in the prefrontal cortex and increased locomotion activity and anxiety levels after exposed to IH, while no change emerged in NOX2 knock-out mice. Treatment of WT mice with the antioxidant/NOX inhibitor apocynin prevented the neuropathological and neurobehavioral alterations induced by IH exposures. Our data suggest that NOX2-derived oxidative stress is involved in the loss of PV-IN in the prefrontal cortex and development of neurobehavioral alterations for adult mice exposed to IH. These results provide a molecular mechanism for the coupling between sleep apnea and brain oxidative stress as well as potential new therapeutic avenues. PMID:25911467

  6. Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

    SciTech Connect

    Tateishi, Yoshihisa Sasabe, Eri; Ueta, Eisaku; Yamamoto, Tetsuya

    2008-02-08

    Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by {gamma}-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

  7. Arctigenin reduces blood pressure by modulation of nitric oxide synthase and NADPH oxidase expression in spontaneously hypertensive rats.

    PubMed

    Liu, Ying; Wang, Guoyuan; Yang, Mingguang; Chen, Haining; Zhao, Yan; Yang, Shucai; Sun, Changhao

    2015-12-25

    Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation. PMID:26585490

  8. Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass.

    PubMed

    Sun, Jinlong; Ming, Leiguo; Shang, Fengqing; Shen, Lijuan; Chen, Jihua; Jin, Yan

    2015-01-01

    Because of the reduced potential for osteogenesis in aging bone marrow stromal cells, the balance of bone metabolism becomes disrupted, leading to various bone diseases. An increase in reactive oxygen species has been determined to be one of the key factors that accelerates the aging process in BMSCs. In these cells, increased expression of NADPH oxidases is the major source of ROS. In the current study, we suppressed the expression of NOX using apocynin, an effective antioxidant and free radical scavenger, and the results showed that aging BMSCs exhibited an enhanced potential for osteogenesis. The expression of potential key targets influencing this reversal was evaluated using qRT-PCR, and the expression of p53 was shown to be reduced with the suppression of NOX. We speculate that this may be one of the major reasons for the reversal of the aging process. We also examined the effect of apocynin in vivo, and the results showed that in SAMP6 mice, bone mineral density and total bone volume were increased after 3 months of apocynin treatment. In conclusion, our results demonstrate that in aging BMSCs, suppression of NADPH oxidase by apocynin partially reverses the aging process and enhances osteogenic potential. PMID:26686764

  9. Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass

    PubMed Central

    Sun, Jinlong; Ming, Leiguo; Shang, Fengqing; Shen, Lijuan; Chen, Jihua; Jin, Yan

    2015-01-01

    Because of the reduced potential for osteogenesis in aging bone marrow stromal cells, the balance of bone metabolism becomes disrupted, leading to various bone diseases. An increase in reactive oxygen species has been determined to be one of the key factors that accelerates the aging process in BMSCs. In these cells, increased expression of NADPH oxidases is the major source of ROS. In the current study, we suppressed the expression of NOX using apocynin, an effective antioxidant and free radical scavenger, and the results showed that aging BMSCs exhibited an enhanced potential for osteogenesis. The expression of potential key targets influencing this reversal was evaluated using qRT-PCR, and the expression of p53 was shown to be reduced with the suppression of NOX. We speculate that this may be one of the major reasons for the reversal of the aging process. We also examined the effect of apocynin in vivo, and the results showed that in SAMP6 mice, bone mineral density and total bone volume were increased after 3 months of apocynin treatment. In conclusion, our results demonstrate that in aging BMSCs, suppression of NADPH oxidase by apocynin partially reverses the aging process and enhances osteogenic potential. PMID:26686764

  10. Curcuminoids Modulate the PKC?/NADPH Oxidase/Reactive Oxygen Species Signaling Pathway and Suppress Matrix Invasion during Monocyte-Macrophage Differentiation.

    PubMed

    Huang, Shao-Lan; Chen, Pei-Yi; Wu, Ming-Jiuan; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-10-14

    Monocyte recruitment and invasion play critical roles in the initiation and progression of atherosclerosis. The reduction in monocyte adhesion and infiltration is thought to exert antiatherosclerotic effects. Curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are the major active components of curcuminoids and exhibit several biological activities, including anti-inflammatory, anticarcinogenic, and hypocholesterolemic activities. The aim of this study was to investigate the antiatherogenic effects and mechanisms of curcuminoids during monocyte to macrophage differentiation. The results showed that curcumin, DMC, and BDMC (20 ?M) suppressed matrix invasion from 100.0 ± 5.0% to 24.8 ± 1.4%, 26.6 ± 2.9%, and 33.7 ± 1.7%, respectively, during PMA-induced THP-1 differentiation. We found that curcuminoids significantly reduced PMA-induced CD11b and MMP-9 expression by THP-1 cells. Production of reactive oxygen species (ROS) induced by PMA (126.7 ± 2.1%) was markedly attenuated by curcumin, DMC, and BDMC to 99.5 ± 7.8%, 87.8 ± 8.2%, and 89.8 ± 7.6%, respectively, resulting in the down-regulation of CD11b and MMP-9 expression. We demonstrated that curcuminoids inhibited NADPH oxidase through the down-regulation of NOX2 expression and the reduction of p47phox membrane translocation. Moreover, we found involvement of PKC? in the PMA-induced NOX2, CD11b, and MMP-9 mRNA expression. Curcumin, DMC, and BDMC decreased the active form of PKC? protein stimulated by PMA in THP-1 cells. Overall, our results reveal that curcuminoids suppress matrix invasion through the inhibition of the PKC?/NADPH oxidase/ROS signaling pathway during monocyte-macrophage differentiation. PMID:26414495

  11. Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase.

    PubMed

    Steffen, Yvonne; Vuillaume, Gregory; Stolle, Katrin; Roewer, Karin; Lietz, Michael; Schueller, Jutta; Lebrun, Stefan; Wallerath, Thomas

    2012-10-15

    The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions ((·)O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation. PMID:22766265

  12. Genipin induces cyclooxygenase-2 expression via NADPH oxidase, MAPKs, AP-1, and NF-?B in RAW 264.7 cells.

    PubMed

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Chung, Young Chul; Kim, Hee Suk; Park, Youn-Joon; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-02-01

    Genipin is a compound found in gardenia fruit extract with diverse pharmacological activities. However, the mechanism underlying genipin-induced cyclooxygenase-2 (COX-2) expression remains unknown. In this study, we investigated the effects of genipin on COX-2 expression and determined that exposure to genipin dose-dependently enhanced the production of prostaglandin E2 (PGE2), a major COX-2 metabolite, in RAW 264.7 cells. These effects were mediated by genipin-induced activation of the COX-2 promoter, as well as AP-1 and NF-?B luciferase constructs. Phosphatidylinositol-3-kinase/Akt and MAPKs were also significantly activated by genipin, and Akt and MAPKs inhibitors (PD98059, SB20358, SP600125, and LY294002) inhibited genipin-induced COX-2 expression. Moreover, genipin increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2 and NOX3. Inhibition of NADPH with diphenyleneiodonium attenuated ROS production, COX-2 expression and NF-?B and AP-1 activation. These results suggest that the molecular mechanism mediating ROS-dependent COX-2 up-regulation and PGE2 production by genipin involves activation of Akt, MAPKs and AP-1/NF-?B. PMID:24296130

  13. Glucose oxidase activity of actinomycetes.

    PubMed

    St Vlahov, S

    1978-01-01

    The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established. PMID:76424

  14. NAD(P)H oxidase and pro-inflammatory response during maximal exercise: role of C242T polymorphism of the P22PHOX subunit.

    PubMed

    Izzicupo, P; Di Valerio, V; D' Amico, M A; Di Mauro, M; Pennelli, A; Falone, S; Alberti, G; Amicarelli, F; Miscia, S; Gallina, S; Di Baldassarre, A

    2010-01-01

    Intense exercise induces a pro-inflammatory status through a mechanism involving the NAD(P)H oxidase system. We focused our attention on p22phox, a subunit of the NAD(P)H oxidase, and on its allelic polymorphism C242T, which is known to affect the functional activity of the enzyme. We investigated whether the p22phox C242T variants exhibit systemic effects in healthy subjects by analyzing the proinflammatory and cardiocirculatory responses to physical exercise in endurance athletes. The group of study consisted of 97 long distance runners, 37 +/- 4.4 yrs of age, with similar training history. The subjects underwent a maximal stress test during which both inflammatory and cardiopulmonary parameters were monitored. Our results demonstrate that T allele deeply influences the neutrophil activation in response to intense exercise, since T carriers were characterized by significantly lower release of myeloperoxidase (MPO), a classical leukocyte derived pro-inflammatory cytokine. In addition, the presence of T allele was associated with a higher cardiopulmonary efficiency as evidenced by a significantly lower Heart Rate (HR) at the peak of exercise and, when a dominant model was assumed, by a higher maximal oxygen uptake (VO2 max). On the other hand, no effects of 242T mutation on the plasmatic total antioxidant capacity (TAC) and on the cortisol responses to the physical exercise were detected. In conclusion, our data support a systemic role for p22phox C242T polymorphism that, modifying the intensity of the inflammatory response, can influence the cardiovascular adaptations elicited by aerobic training. These results contribute to support the hypothesis of a systemic effect for the C242T polymorphism and of its possible functional rebound in healthy subjects. PMID:20378006

  15. Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells

    SciTech Connect

    Manea, Adrian; Tanase, Laurentia I.; Raicu, Monica; Simionescu, Maya

    2010-06-11

    Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

  16. Glycated albumin triggers fibrosis and apoptosis via an NADPH oxidase/Nox4-MAPK pathway-dependent mechanism in renal proximal tubular cells.

    PubMed

    Qi, Weiwei; Niu, Jianying; Qin, Qiaojing; Qiao, Zhongdong; Gu, Yong

    2015-04-15

    Glycated albumin (GA), an Amadori product used as a marker of hyperglycemia and the early-stage glycation products compared to AGEs, might further promote kidney lesions in diabetic nephropathy (DN). However, the mechanisms how GA cause proximal tubular cells damage remain poorly understood. In this study, we investigated the effects of GA on fibrosis and apoptosis of renal proximal tubular cells (NRK-52E) in vitro experiments. Our results showed that GA promoted ?-SMA, fibronectin (FN) and TGF-? expressions in NRK-52E cells. GA also increased cell apoptosis and stimulated the expressions of pro-caspase 3/cleaved-caspase 3. GA overloading enhanced the phosphorylation of MAPK pathway. GA-induced ?-SMA, FN, TGF-? and caspase 3 expressions were completely suppressed by the NADPH oxidase inhibitor apocynin (Apo), the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the latent antioxidant Astragaloside IV (AS-IV). Real-time PCR showed that GA increased Nox1, Nox2 and Nox4 mRNA expressions, especially the Nox4 expression. Furthermore, Nox4 siRNA blocked GA-induced tubular damages and the MAPK pathway activation. These results demonstrate that GA increases the permissiveness of proximal tubular cells to fibrosis and apoptosis in vitro by triggering a pathway that involves NADPH oxidase/Nox4-MAPK signaling pathway. This event may represent a key cellular effect in increasing the susceptibility of tubular cells to fibrosis and apoptosis when the tubules cope with a high GA load. This effect is instrumental to renal damage and disease progression in patients with DN. PMID:25681565

  17. Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes

    SciTech Connect

    Shvedova, A.A. Kisin, E.R.; Murray, A.R.; Kommineni, C.; Castranova, V.; Fadeel, B.; Kagan, V.E.

    2008-09-01

    Single-walled carbon nanotubes (SWCNT) have been introduced into a large number of new technologies and consumer products. The combination of their exceptional features with very broad applications raised concerns regarding their potential health effects. The prime target for SWCNT toxicity is believed to be the lung where exposure may occur through inhalation, particularly in occupational settings. Our previous work has demonstrated that SWCNT cause robust inflammatory responses in rodents with very early termination of the acute phase and rapid onset of chronic fibrosis. Timely elimination of polymorphonuclear neutrophils (PMNs) through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. Thus, we hypothesized that NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. To experimentally address the hypothesis, we employed NADPH oxidase-deficient mice which lack the gp91{sup phox} subunit of the enzymatic complex. We found that NADPH oxidase null mice responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-{beta}, and significantly lower levels of collagen deposition, as compared to C57BL/6 control mice. These results demonstrate a role for NADPH oxidase-derived reactive oxygen species in determining course of pulmonary response to SWCNT.

  18. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  19. NDS27 combines the effect of curcumin lysinate and hydroxypropyl-?-cyclodextrin to inhibit equine PKC? and NADPH oxidase involved in the oxidative burst of neutrophils

    PubMed Central

    Derochette, Sandrine; Mouithys-Mickalad, Ange; Franck, Thierry; Collienne, Simon; Ceusters, Justine; Deby-Dupont, Ginette; Neven, Philippe; Serteyn, Didier

    2014-01-01

    Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of inflammatory diseases. After appropriate stimuli, protein kinase C (PKC) triggers the assembly of NADPH oxidase (Nox2) which produces superoxide anion (O2•?), from which ROS derive. The therapeutic use of polyphenols is proposed to lower ROS production by limiting Nox2 and PKC activities. The purpose of this study was to compare the antioxidant effect of NDS27 and NDS28, two water-soluble forms of curcumin lysinate respectively complexed with hydroxypropyl-?-cyclodextrin (HP?CD) and ?-cyclodextrin (?-CD), on the activity of Nox2 and PKC?, involved in the Nox2 activation pathway. Our results, showed that NDS27 is the best inhibitor for Nox2 and PKC?. This was illustrated by the combined effect of HP?CD and curcumin lysinate: HP?CD, but not ?-CD, improved the release of curcumin lysinate and its exchange against lipid or cholesterol as demonstrated by the lipid colouration with Oil Red O, the extraction of radical lipophilic probes recorded by ESR and the HPLC measurements of curcumin. HP?CD not only solubilised and transported curcumin, but also indirectly enhanced its action on both PKC and Nox2 activities. The modulatory effect of NDS27 on the Nox2 activation pathway of neutrophils may open therapeutic perspectives for the control of pathologies with excessive inflammatory reactions. PMID:25493216

  20. Activation of Polyphenol Oxidase of Chloroplasts 1

    PubMed Central

    Tolbert, N. E.

    1973-01-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or —18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density. Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles × mg?1 chlorophyll × hr?1. Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes. Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30 minutes. No single peptide from the digested trypsin was found to be the sole activating factor. About 0.25 ?g of trypsin activated 50% the polyphenol oxidase activity in a standard chloroplast assay containing 2.1 ?g of chlorophyll. Treatment of spinach chloroplasts with tris buffer or ethylenediamine tetraacetate extracted the ATPase activity, but the polyphenol oxidase activity remained with the broken plastids. However these treatments increased the latent polyphenol oxidase activity 50- to 100-fold. Chloroplasts from a second group of plants, including alfalfa, wheat, oats, peas, and sugarcane leaves, oxidized dihydroxyphenylalanine at a rate of 11 to 120 ?moles × mg?1 chlorophyll × hr?1. Polyphenol oxidase in these chloroplasts required a low intensity of red light for activity. Fifty or 75% activation of the oxidase in wheat chloroplasts required 4 to 6 foot candles of light and more light was required for alfalfa chloroplasts. Blue or far red light were ineffective. Trypsin was inhibitory. Upon aging chloroplasts from wheat leaves, but not alfalfa or peas, for 5 to 7 days at 4 C the total polyphenol oxidase activity did not increase, but the activation characteristics changed to those of chloroplasts from the spinach group. Chloroplasts from a third group of plants, including bean, tomato, and corn leaves, slowly oxidized dihydroxyphenylalanine in the dark and exhibited no latency. PMID:16658308

  1. Diapocynin, a Dimer of the NADPH Oxidase Inhibitor Apocynin, Reduces ROS Production and Prevents Force Loss in Eccentrically Contracting Dystrophic Muscle

    PubMed Central

    Ismail, Hesham M.; Scapozza, Leonardo; Ruegg, Urs T.; Dorchies, Olivier M.

    2014-01-01

    Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches. PMID:25329652

  2. Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba

    PubMed Central

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

  3. FgNoxR, a regulatory subunit of NADPH oxidases, is required for female fertility and pathogenicity in Fusarium graminearum.

    PubMed

    Zhang, Chengkang; Lin, Yahong; Wang, Jianqiang; Wang, Yang; Chen, Miaoping; Norvienyeku, Justice; Li, Guangpu; Yu, Wenying; Wang, Zonghua

    2016-01-01

    Fusarium graminearum is a filamentous fungal pathogen that causes wheat Fusarium head blight. In this study, we identified FgNoxR, a regulatory subunit of NADPH oxidases (Nox) in F. graminearum, and found that it plays an important role in the pathogenicity of F. graminearum. FgNoxR is localized on punctate structures throughout the cytoplasm in aerial hyphae while these structures tend to accumulate at or near the plasma membrane, septa and hyphal tips in germinated conidia. Deletion of the FgNOXR gene results in reduced conidiation and germination. Importantly, sexual development is totally abolished in the FgNOXR deletion mutant. In addition, the disease lesion of FgNOXR deletion mutant is limited to the inoculated spikelets of wheat heads. Finally, FgNoxR interacts with FgRac1 and FgNoxA, and all three proteins are required for female fertility. Taken together, our data indicate that FgNoxR contributes to conidiation, sexual reproduction and pathogenesis in F. graminearum. PMID:26607286

  4. The regulation of methyl jasmonate on hyphal branching and GA biosynthesis in Ganoderma lucidum partly via ROS generated by NADPH oxidase.

    PubMed

    Shi, Liang; Gong, Li; Zhang, Xiangyang; Ren, Ang; Gao, Tan; Zhao, Mingwen

    2015-08-01

    Ganoderma lucidum is one of the best known medicinal basidiomycetes because it produces many pharmacologically active compounds, and methyl jasmonate (MeJA) was previously reported to induce the biosynthesis of ganoderic acids (GA) in G. lucidum. In this study, we found that MeJA not only increased the amount of GA but also increased the distance between hyphal branches by approximately 1.2-fold. Further analysis showed that MeJA could increase the intracellular ROS (reactive oxygen species) content by approximately 2.2-2.7-fold. Furthermore, the hyphal branching and GA biosynthesis regulated by MeJA treatment could be abolished by ROS scavengers to a level similar to or lower than that of the control group. These results indicated that the regulation of hyphal branching and GA biosynthesis by MeJA might occur via a ROS signaling pathway. Further analysis revealed that NADPH oxidase (NOX) plays an important role in MeJA-regulated ROS generation. Importantly, our results highlight that NOX functions in signaling cross-talk between ROS and MeJA. In addition, these findings provide an excellent opportunity to identify potential pathways linking ROS networks to MeJA signaling in fungi and suggest that plants and fungi share a conserved signaling-crosstalk mechanism. PMID:25512263

  5. The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines.

    PubMed

    Carnesecchi, S; Rougemont, A-L; Doroshow, J H; Nagy, M; Mouche, S; Gumy-Pause, F; Szanto, I

    2015-07-01

    Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK(+) ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK(+) ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK(+) ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK(+) ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK(+) ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK(+) ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK(+) ALCL patients that resist classical therapeutic approaches. PMID:25797883

  6. H2O2 generated by NADPH oxidase 4 contributes to transient receptor potential vanilloid 1 channel-mediated mechanosensation in the rat kidney.

    PubMed

    Lin, Chian-Shiung; Lee, Shang-Hsing; Huang, Ho-Shiang; Chen, Yih-Sharng; Ma, Ming-Chieh

    2015-08-15

    The presence of NADPH oxidase (Nox) in the kidney, especially Nox4, results in H2O2 production, which regulates Na(+) excretion and urine formation. Redox-sensitive transient receptor potential vanilloid 1 channels (TRPV1s) are distributed in mechanosensory fibers of the renal pelvis and monitor changes in intrapelvic pressure (IPP) during urine formation. The present study tested whether H2O2 derived from Nox4 affects TRPV1 function in renal sensory responses. Perfusion of H2O2 into the renal pelvis dose dependently increased afferent renal nerve activity and substance P (SP) release. These responses were attenuated by cotreatment with catalase or TRPV1 blockers. In single unit recordings, H2O2 activated afferent renal nerve activity in response to rising IPP but not high salt. Western blots revealed that Nox2 (gp91(phox)) and Nox4 are both present in the rat kidney, but Nox4 is abundant in the renal pelvis and originates from dorsal root ganglia. This distribution was associated with expression of the Nox4 regulators p22(phox) and polymerase ?-interacting protein 2. Coimmunoprecipitation experiments showed that IPP increases polymerase ?-interacting protein 2 association with Nox4 or p22(phox) in the renal pelvis. Interestingly, immunofluorescence labeling demonstrated that Nox4 colocalizes with TRPV1 in sensory fibers of the renal pelvis, indicating that H2O2 generated from Nox4 may affect TRPV1 activity. Stepwise increases in IPP and saline loading resulted in H2O2 and SP release, sensory activation, diuresis, and natriuresis. These effects, however, were remarkably attenuated by Nox inhibition. Overall, these results suggest that Nox4-positive fibers liberate H2O2 after mechanostimulation, thereby contributing to a renal sensory nerve-mediated diuretic/natriuretic response. PMID:26136558

  7. The Involvement of the Tyrosine Kinase c-Src in the Regulation of Reactive Oxygen Species Generation Mediated by NADPH Oxidase-1

    PubMed Central

    Gianni, Davide; Bohl, Ben; Courtneidge, Sara A.

    2008-01-01

    NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers. PMID:18463161

  8. Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion

    NASA Technical Reports Server (NTRS)

    Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.; Jo, Hanjoong

    2003-01-01

    Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

  9. Apocyanin, NADPH oxidase inhibitor prevents lipopolysaccharide induced ?-synuclein aggregation and ameliorates motor function deficits in rats: Possible role of biochemical and inflammatory alterations.

    PubMed

    Sharma, Neha; Kapoor, Monika; Nehru, Bimla

    2016-01-01

    Parkinson's disease (PD), is an age-related, progressive neurodegenerative disorder that affects movement and is characterized by the loss of dopaminergic neurons in the nigrostriatal region. Although the clinical and pathological features of PD are complex, recent studies have indicated that microglial NADPH oxidase play a key role in its pathology. A little information is available regarding the role of apocyanin, an NADPH oxidase inhibitor, in ameliorating ?-synuclein aggregation and neurobehavioral consequences of PD. Therefore, the present study evaluated its therapeutic potentials for the treatment of neurobehavioral consequences in lipolysaccharide (LPS) induced PD model. For the establishment of PD model LPS (5?g/5?l PBS) was injected into the Substantia nigra (SN) of rats. Apocyanin (10mg/kgb.wt) was injected intraperitoneal. Statistical analysis revealed that apocynin significantly ameliorated LPS induced inflammatory response characterized by NFkB, TNF-? and IL-1? upregulation as assessed by ELISA. It also prevented dopaminergic neurons from toxic insult of LPS as indicated by inhibition of apoptotic markers i.e., caspase 3 and caspase 9 as depicted from RT-PCR and ELISA studies. This was further supported by TUNEL assay for DNA fragmentation. Effectiveness of apocyanin in protecting dopaminergic neuronal degeneration was further confirmed by assessment of ?-synuclein deposition as depicted by IHC analysis. Consequently, an improvement in the behavioral outcome was observed following apocyanin treatment as depicted from various behavioral tests performed. Hence the data suggests that specific NADPH oxidase inhibitors, such as apocynin, may provide a new therapeutic approach to the control of neurological disabilities induced by LPS induced PD. PMID:26367469

  10. Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion.

    PubMed

    Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P; McNally, J Scott; Holland, Steven M; Dikalov, Sergei; Giddens, Don P; Griendling, Kathy K; Harrison, David G; Jo, Hanjoong

    2003-11-21

    Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event. PMID:12958309

  11. NADPH oxidase NOX5-S and nuclear factor ?B1 mediate acid-induced microsomal prostaglandin E synthase-1 expression in Barrett's esophageal adenocarcinoma cells.

    PubMed

    Zhou, Xiaoxu; Li, Dan; Resnick, Murray B; Wands, Jack; Cao, Weibiao

    2013-05-01

    The mechanisms of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not known. Cycloxygenase-2 (COX-2)-derived prostaglandin E? (PGE?) has been shown to be important in esophageal tumorigenesis. We have shown that COX-2 mediates acid-induced PGE? production. The prostaglandin E synthase (PGES) responsible for acid-induced PGE2 production in BE, however, is not known. We found that microsomal PGES1 (mPGES1), mPGES2, and cytosolic PGES (cPGES) were present in FLO EA cells. Pulsed acid treatment significantly increased mPGES1 mRNA and protein levels but had little or no effect on mPGES2 or cPGES mRNA. Knockdown of mPGES1 by mPGES1 small interfering RNA (siRNA) blocked acid-induced increase in PGE2 production and thymidine incorporation. Knockdown of NADPH oxidase, NOX5-S, a variant lacking calcium-binding domains, by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a nuclear factor ?B (NF-?B) in vivo activation reporter plasmid pNF-?B-Luc. Knockdown of NF-?B1 p50 by p50 siRNA significantly decreased acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE? production. Two novel NF-?B binding elements, GGAGTCTCCC and CGGGACACCC, were identified in the mPGES1 gene promoter. We conclude that mPGES1 mediates acid-induced increase in PGE? production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-?B1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. PMID:23439561

  12. Rosuvastatin treatment protects against nitrate-induced oxidative stress in eNOS knockout mice: implication of the NAD(P)H oxidase pathway

    PubMed Central

    Otto, Anne; Fontaine, Jeanine; Tschirhart, Eric; Fontaine, David; Berkenboom, Guy

    2006-01-01

    Nitrate tolerance is associated with an enhanced superoxide anion (O2?) production and may be attenuated by statins as they interact with the two main endothelial NO synthase (eNOS) and NAD(P)H oxidase pathways involved in this oxidative stress. Groups of wild-type (wt, C57Bl/6J) and eNOS knock-out mice (eNOS?/?) received rosuvastatin (20?mg?kg?1?day?1 p.o.) for 5 weeks and a cotreatment with the statin plus nitroglycerin (NTG; 30?mg?kg?1?day?1, subcutaneous injections b.i.d.) for the last 4 days. Another group received only NTG (30?mg?kg?1?d?1, b.i.d. for 4 days) and finally control mice from both strains received no treatment. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1?nM–0.1?mM) were determined on thromboxane analogue (U44619)-precontracted rings and O2? production (RLU 5?s?1?mg?1 of total protein content) was assessed in aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Reverse transcriptase–polymerase chain reaction analysis was performed on aortas from both mice strains. In vivo NTG treatment induced a significant rightward shift of the concentration–effect curve to NTG compared to control group. There was, however, no cross-tolerance with non-nitrate sources of NO (unaltered response to acetylcholine in wt group). The rosuvastatin+NTG cotreatment was able to protect against the development of nitrate tolerance in both mice strains and L-mevalonate abolished this protective effect of rosuvastatin. In vivo treatment with apocynin, a purported NAD(P)H oxidase inhibitor, also produced a similar protection to that observed with rosuvastatin in both strains. Superoxide anion formation was increased after NTG treatment in both mice strains and the rosuvastatin+NTG cotreatment was able to reduce that production. Moreover, rosuvastatin treatment abolished the increase in gp91phox mRNA (an endothelial membrane NAD(P)H oxidase subunit) expression induced by in vivo exposure to NTG. These findings suggest that long-term rosuvastatin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O2? production, probably via a direct interaction with the NAD(P)H oxidase pathway. PMID:16633368

  13. Effects of oximes on mitochondrial oxidase activity.

    PubMed

    Sakurada, Koichi; Ikegaya, Hiroshi; Ohta, Hikoto; Fukushima, Hisayo; Akutsu, Tomoko; Watanabe, Ken

    2009-09-10

    Oximes, including 2-pyridinealdoxime methiodide (2-PAM), are reactivators of acetylcholinesterase (AChE) inhibited by organophosphate poisoning. Unfortunately, their clinical use has been limited by their toxicity. To investigate the mechanism of this toxicity, the effects of oximes on the enzymes choline oxidase (ChOD) and cytochrome c oxidase (CyCOD) of the respiratory chain in mitochondria were examined. The oximes 2-PAM, obidoxime, and diacetylmonoxime significantly (P<0.01) inhibited ChOD activity, and the extent of inhibition correlated with the ability to reactivate inhibited AChE. When ChOD activity in mitochondrial extracts was tested, 2-PAM inhibited the activity by 75%, obidoxime and diacetylmonoxime did not significantly inhibit it, and 4-[(hydroxy-imino)methyl]-1-decylpyridinium bromide (4-PAD), which has greater toxicity, increased the amount of product generated in the assay to approximately 200% of normal levels. Similarly, 2-PAM inhibited the activity of CyCOD in mitochondrial extracts whereas obidoxime and diacetylmonoxime did not. One explanation for these findings is that, in addition to their inhibition of mitochondrial oxidases, the oximes may produce excessive reactive oxygen species such as H(2)O(2) in the mitochondrial fraction, which may account for some of their toxicity. This is a preliminary report related to the toxicities of oximes that may participate in the inactivation of mitochondrial oxidase enzymes. This hypothesis should be further investigated by in vivo study, including kinetic determination and free radical work. PMID:19465093

  14. ZmMPK5 is required for the NADPH oxidase-mediated self-propagation of apoplastic H2O2 in brassinosteroid-induced antioxidant defence in leaves of maize

    PubMed Central

    Zhang, Aying; Zhang, Jun; Ye, Nenghui; Cao, Jianmei; Tan, Mingpu; Zhang, Jianhua; Jiang, Mingyi

    2010-01-01

    Brassinosteroids (BRs) have been shown to induce hydrogen peroxide (H2O2) accumulation, and BR-induced H2O2 up-regulates antioxidant defence systems in plants. However, the mechanisms by which BR-induced H2O2 regulates antioxidant defence systems in plants remain to be determined. In the present study, the role of ZmMPK5, a mitogen-activated protein kinase, in BR-induced anitioxidant defence and the relationship between the activation of ZmMPK5 and H2O2 production in BR signalling were investigated in leaves of maize (Zea mays) plants. BR treatment activated ZmMPK5, induced apoplastic and chloroplastic H2O2 accumulation, and enhanced the total activities of antioxidant enzymes. Such enhancements were blocked by pre-treatment with mitogen-activated protein kinase kinase (MAPKK) inhibitors and H2O2 inhibitors or scavengers. Pre-treatment with MAPKK inhibitors substantially arrested the BR-induced apoplastic H2O2 production after 6?h of BR treatment, but did not affect the levels of apoplastic H2O2 within 1?h of BR treatment. BR-induced gene expression of NADPH oxidase was also blocked by pre-treatment with MAPKK inhibitors and an apoplastic H2O2 inhibitor or scavenger after 120?min of BR treatment, but was not affected within 30?min of BR treatment. These results suggest that the BR-induced initial apoplastic H2O2 production activates ZmMPK5, which is involved in self-propagation of apoplastic H2O2 via regulation of NADPH oxidase gene expression in BR-induced antioxidant defence systems. PMID:20693409

  15. Developmental loss of parvalbumin-positive cells in the prefrontal cortex and psychiatric anxiety after intermittent hypoxia exposures in neonatal rats might be mediated by NADPH oxidase-2.

    PubMed

    Liang, Dong; Li, Guowei; Liao, Xingzhi; Yu, Dawei; Wu, Jing; Zhang, Mingqiang

    2016-01-01

    Sleep apnea is more frequently experienced in neonatal life. Here we investigated the causal contribution of NOX2-derived oxidative stress in the prefrontal cortex (PFC) to neurodevelopmental alterations and psychiatric anxiety in a neonatal rat model of sleep apnea. Neonatal postnatal day 5 (P5) rats were exposed to long-term intermittent hypoxia (LTIH) or room air (RA) for 10 days. In the PFC, we determined the impact (I) of LTIH exposures on NADPH oxidase-2 (NOX2) expression and oxidative stress (II) of pharmacological NOX2 inhibition on LTIH-induced neurodevelopmental alterations in the P14 and P49 rats. Endpoints were NOX2-derived oxidative stress, parvalbumin (PV)-positive cells (PV-cells) and psychiatric anxiety. The results showed neonatal LTIH exposures increased NOX2 expression in the PFC of P14 rats, which was accompanied with elevation of NOX activity. Neonatal LTIH exposures increased oxidative stress in cortical PV-cells characterized by elevation of 8-hydroxy-20-deoxyguanosine (8-OHDG) level and reduced PV immunoreactivity, PV-cell counts in the PFC of P14 and P49 rats. Neonatal LTIH exposures increased psychiatric anxiety levels in the P49 rats. Pretreatment of neonatal rats before each neonatal LTIH exposure with the antioxidant/NOX inhibitor apocynin prevented the reduced PV immunoreactivity, PV-cells loss in the PFC and development of anxiety-like behavior. Our data suggest that NOX2-derived oxidative stress might be involved in the developmental loss of PV-cells in the PFC and development of psychiatric anxiety for neonatal rats exposed to LTIH. PMID:26319087

  16. Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase

    PubMed Central

    Cozzoli, Anna; Liantonio, Antonella; Conte, Elena; Cannone, Maria; Massari, Ada Maria; Giustino, Arcangela; Scaramuzzi, Antonia; Pierno, Sabata; Mantuano, Paola; Capogrosso, Roberta Francesca; Camerino, Giulia Maria

    2014-01-01

    Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 ?M) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers. PMID:25080489

  17. Investigation of the C242T polymorphism of NAD(P)H oxidase p22 phox gene and ischaemic heart disease using family-based association methods.

    PubMed

    Spence, M S; McGlinchey, P G; Patterson, C C; Allen, A R; Murphy, G; Bayraktutan, U; Fogarty, D G; Evans, A E; McKeown, P P

    2003-12-01

    Ischaemic heart disease is a complex phenotype arising from the interaction of genetic and environmental factors. Excessive production of reactive oxygen species leading to endothelial dysfunction is believed to be important in the pathogenesis of ischaemic heart disease. The NAD(P)H oxidase system generates superoxide anions in vascular cells; however, the role of the C242T polymorphism of the NAD(P)H oxidase p22 phox gene in ischaemic heart disease is unclear due to contradictory results from case-control studies. Consequently, we applied family-based association tests to investigate the role of this polymorphism in ischaemic heart disease in a well-defined Irish population. A total of 1023 individuals from 388 families (discordant sibships and parent/child trios) were recruited. Linkage disequilibrium between the polymorphism and ischaemic heart disease was tested using the combined transmission disequilibrium test (TDT)/sib-TDT (cTDT) and pedigree disequilibrium test (PDT). Both cTDT and PDT analyses found no statistically significant excess transmission of either allele to affected individuals (P =0.30 and P =0.28, respectively). Using robust family-based association tests specifically designed for the study of complex diseases, we found no evidence that the C242T polymorphism of the p22 phox gene has a significant role in the development of ischaemic heart disease in our population. PMID:12877653

  18. Reactive oxygen species produced by NADPH oxidase and mitochondrial dysfunction in lung after an acute exposure to Residual Oil Fly Ashes

    SciTech Connect

    Magnani, Natalia D.; Marchini, Timoteo; Vanasco, Virginia; Tasat, Deborah R.; Alvarez, Silvia; Evelson, Pablo

    2013-07-01

    Reactive O{sub 2} species production triggered by particulate matter (PM) exposure is able to initiate oxidative damage mechanisms, which are postulated as responsible for increased morbidity along with the aggravation of respiratory diseases. The aim of this work was to quantitatively analyse the major sources of reactive O{sub 2} species involved in lung O{sub 2} metabolism after an acute exposure to Residual Oil Fly Ashes (ROFAs). Mice were intranasally instilled with a ROFA suspension (1.0 mg/kg body weight), and lung samples were analysed 1 h after instillation. Tissue O{sub 2} consumption and NADPH oxidase (Nox) activity were evaluated in tissue homogenates. Mitochondrial respiration, respiratory chain complexes activity, H{sub 2}O{sub 2} and ATP production rates, mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria. ROFA exposure was found to be associated with 61% increased tissue O{sub 2} consumption, a 30% increase in Nox activity, a 33% increased state 3 mitochondrial O{sub 2} consumption and a mitochondrial complex II activity increased by 25%. During mitochondrial active respiration, mitochondrial depolarization and a 53% decreased ATP production rate were observed. Neither changes in H{sub 2}O{sub 2} production rate, nor oxidative damage in isolated mitochondria were observed after the instillation. After an acute ROFA exposure, increased tissue O{sub 2} consumption may account for an augmented Nox activity, causing an increased O{sub 2}{sup ·?} production. The mitochondrial function modifications found may prevent oxidative damage within the organelle. These findings provide new insights to the understanding of the mechanisms involving reactive O{sub 2} species production in the lung triggered by ROFA exposure. - Highlights: • Exposure to ROFA alters the oxidative metabolism in mice lung. • The augmented Nox activity contributes to the high tissue O{sub 2} consumption. • Exposure to ROFA produces alterations in mitochondrial function. • ??{sub m} decrease in state 3 may be responsible for the decreased ATP production. • Mild uncoupling prevents mitochondrial oxidative damage.

  19. Structural Assembly of the Active Site in an Aldo-keto Reductase by NADPH Cofactor

    E-print Network

    Blaber, Michael

    , Tallahassee FL 32306-4380, USA A 1.9 AÊ resolution X-ray structure of the apo-form of Corynebacterium 2; NADPH; allostery; active site; catalysis*Corresponding author Introduction Corynebacterium 2,5-diketo

  20. Focal Ischemic Injury with Complex Middle Cerebral Artery in Stroke-Prone Spontaneously Hypertensive Rats with Loss-Of-Function in NADPH Oxidases

    PubMed Central

    Yao, Hiroshi; Ferdaus, Mohammed Zubaerul; Zahid, Hasan Md.; Ohara, Hiroki; Nakahara, Tatsuo; Nabika, Toru

    2015-01-01

    By means of introgressing a loss-of-function mutation in the p22phox gene from the Matsumoto Eosinophilia Shinshu (MES) rat to stroke-prone spontaneously hypertensive rats (SHRSP), we constructed the SHRSP-based congenic strain lacking the P22PHOX expression (i.e., lacking NADPH oxidases [NOX] activities) (SHRSP.MES-Cybames/Izm; hereafter referred to as SP.MES). To examine the effects of Nox activities on the focal ischemic injury or stroke, we performed middle cerebral artery (MCA) occlusion in this new congenic strain; the distal MCA was occluded by 561-nm laser-driven photothrombosis. Resting mean arterial blood pressure was significantly lower in SP.MES when compared with the control PM0/SHRSP (150±11 mmHg vs. 166±11 mmHg). Cerebral blood flow decreased to 37±13% in SP.MES and 35±17% in PM0/SHRSP at 10 min after MCA occlusion (not significant). Infarct volume determined at 24 h after MCA occlusion in SP.MES was 89±39 mm3, which was not significantly different from 83±35 mm3 in PM0/SHRSP. The distal MCA pattern was more complex in SP.MES (median 3, IQR 3–5) than PM0/SHRSP (median 2, IQR 1–3) (p = 0.001). Because more complex distal MCA is known to produce larger infarction after distal MCA occlusion in SHR, we adjusted for the branching pattern in an ANCOVA. The adjusted mean of infarct volume was significantly smaller in SP.MES compared with that in PM0/SHRSP (67 [95% CI 46 to 87] mm3 vs. 100 [95% CI 82 to 118] mm3, p = 0.032). Elimination of the P22PHOX expression induced complex distal MCA, which would suggest the presence of ‘loss of complexity’ induced by enhanced oxidative stress in SHRSP; infarct size in SP.MES—when adjusted for distal MCA complexity—was significantly attenuated compared with that in PM0/SHRSP. Therefore, the present results suggest that Nox is harmful for ischemic brain tissue. PMID:26389812

  1. Ramipril treatment protects against nitrate-induced oxidative stress in eNOS-/- mice: An implication of the NADPH oxidase pathway.

    PubMed

    Otto, Anne; Fontaine, Jeanine; Berkenboom, Guy

    2006-07-01

    The development of nitrate tolerance has been found to be associated with vascular production of superoxide anion (O2-*), generated mainly by the eNOS and NADPH oxidase pathways. The aim of our study was to investigate whether long-term angiotensin-converting enzyme inhibition by ramipril is able to protect against nitrate tolerance in the aortas of eNOS-deficient (eNOS-/-) mice and to assess the implication of the NADPH oxidase pathway. Therefore, 3 types of treatment were given to wild-type (WT) and eNOS-/- mice: group 1 received ramipril for 5 weeks and a co-treatment with ramirpil plus nitroglycerine (NTG) during the last 4 days, group 2 received only NTG, and group 3 served as control. Relaxations to NTG (0.1 nmol/L to 0.1 mmol/L) were determined on U44619, a thromboxane analogue, precontracted rings, and O2-* production were assessed on aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Cyclic guanosine monophosphate and reverse-transcriptase-polymerase chain reaction analyses were performed on whole mouse aortas. In WT group 2, the concentration-effect curves to NTG were significantly shifted to the right: the pD2 was 6.16 +/- 0.17 (n = 6) vs 6.81 +/- 0.10 (n = 6) in WT group 3 (not exposed to NTG; P < 0.05) and O2-* production was enhanced from 100% +/- 11% (n = 9) to 191% +/- 21% (n = 6; P < 0.01). In contrast, in WT group 1, the rightward shift was abolished: the pD2 value was 6.73 +/- 0.13 (n = 6; NS vs group 3 WT) and O2-* production was 117% +/- 6% (n = 7; NS vs group 3 WT). In eNOS groups 1 and 3, similar data were observed: the pD2 values were 7.58 +/- 0.08 and 7.38 +/- 0.11 (NS) vs 6.89 +/- 0.20 in eNOS group 2 (n = 6; P < 0.01). In the WT mice aortas, ramipril treatment significantly increased the cyclic guanosine monophosphate levels (reflecting nitric oxide availability), which returned to control values after in vivo co-treatment with a bradykinin BK2 antagonist (Icatibant). In both strains, candesartan, an AT1 blocker, was also able to protect against the development of nitrate tolerance. Moreover, before NTG exposure, ramipril treatment decreased p22phox and gp91phox (essential NADPH oxidase subunits) mRNA expression in aortas from both mice strains. In conclusion, long-term ramipril treatment in mice protects against the development of nitrate tolerance by counteracting NTG-induced increase in O2 production, which involves a direct interaction with the NADPH oxidase pathway and seems to be completely independent of the eNOS pathway. PMID:16891913

  2. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase.

    PubMed

    Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro; Kobayashi, Yuji; Kawahara, Kazuki; Yoshida, Takuya; Ohkubo, Tadayasu

    2015-12-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D2 (PGD2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP(+), and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. PMID:26518650

  3. Tomato SlRbohB, a member of the NADPH oxidase family, is required for disease resistance against Botrytis cinerea and tolerance to drought stress

    PubMed Central

    Li, Xiaohui; Zhang, Huijuan; Tian, Limei; Huang, Lei; Liu, Shixia; Li, Dayong; Song, Fengming

    2015-01-01

    NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs) are key enzymes that catalyze the generation of reactive oxygen species (ROS) in plants. In the present study, eight SlRboh genes were identified in tomato and their possible involvement in resistance to Botrytis cinerea and drought tolerance was examined. Expression of SlRbohs was induced by B. cinerea and Pseudomonas syringae pv. tomato but displayed distinct patterns. Virus-induced gene silencing based silencing of SlRbohB resulted in reduced resistance to B. cinerea but silencing of other SlRbohs did not affect the resistance. Compared to non-silenced plants, the SlRbohB-silenced plants accumulated more ROS and displayed attenuated expression of defense genes after infection with B. cinerea. Silencing of SlRbohB also suppressed flg22-induced ROS burst and the expression of SlLrr22, a marker gene related to PAMP-triggered immunity (PTI). Transient expression of SlRbohB in Nicotiana benthamiana led to enhanced resistance to B. cinerea. Furthermore, silencing of SlRbohB resulted in decreased drought tolerance, accelerated water loss in leaves and the altered expression of drought-responsive genes. Our data demonstrate that SlRbohB positively regulates the resistance to B. cinerea, flg22-induced PTI, and drought tolerance in tomato. PMID:26157450

  4. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  5. Effect of exenatide on the cardiac expression of adiponectin receptor 1 and NADPH oxidase subunits and heart function in streptozotocin-induced diabetic rats

    PubMed Central

    2014-01-01

    Background This study investigated the effect of exenatide on the cardiac expression of adiponectin receptor 1 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and heart function in streptozotocin-induced diabetic rats. Methods Male Sprague–Dawley rats were randomly divided into four groups, i.e. control group, diabetic group, diabetic treated with low doses of exenatide (2 ?g?·?kg?1.d?1) and diabetic treated with high doses of exenatide (10 ?g?·?kg?1.d?1). Diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg body weight). At the termination after exenatide treatment for eight weeks, following anesthesia of the rats, a catheter was inserted into the left ventricle through the right common carotid artery for measurement of left ventricular pressure, which included left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the maximal rate of rise and decline of ventricular pressure (±dp/dt[max]). Plasma and myocardial adiponectin levels, and the expressions of myocardial adiponectin receptor 1, p22phox, NADPH oxidase 4 (NOX4), glucose transporter type 4 (Glut4), AMPK-?, phosphorylated-AMPK-?, connective tissue growth factor (CTGF) and copper zinc superoxide dismutase (Cu-Zn-SOD) were assayed. Results Heart function, plasma adiponectin levels, the protein expression of myocardial phosphorylated-AMPK-?, the mRNA expression of myocardial Glut4, and the positive expression of myocardial Cu-Zn-SOD were significantly decreased in diabetic. The protein expression of myocardial adiponectin receptor 1, the mRNA expression of myocardial p22phox and NOX4, and the positive expression of myocardial CTGF were significantly increased in diabetic. Low and high doses of exenatide treatment significantly attenuated these changes in diabetic rats. Conclusions These results suggest that exenatide may contribute to the improvement of the heart function in diabetic rats by down-regulating the expression of myocardial adiponectin receptor 1, p22phox and NOX4, and up-regulating plasma adiponectin level and the expression of myocardial AMPK-?, Glut4 and Cu-Zn-SOD. PMID:24576329

  6. Activated barrier crossing dynamics in the non-radiative decay of NADH and NADPH

    NASA Astrophysics Data System (ADS)

    Blacker, Thomas S.; Marsh, Richard J.; Duchen, Michael R.; Bain, Angus J.

    2013-08-01

    In live tissue, alterations in metabolism induce changes in the fluorescence decay of the biological coenzyme NAD(P)H, the mechanism of which is not well understood. In this work, the fluorescence and anisotropy decay dynamics of NADH and NADPH were investigated as a function of viscosity in a range of water-glycerol solutions. The viscosity dependence of the non-radiative decay is well described by Kramers and Kramers-Hubbard models of activated barrier crossing over a wide viscosity range. Our combined lifetime and anisotropy analysis indicates common mechanisms of non-radiative relaxation in the two emitting states (conformations) of both molecules. The low frequencies associated with barrier crossing suggest that non-radiative decay is mediated by small scale motion (e.g. puckering) of the nicotinamide ring. Variations in the fluorescence lifetimes of NADH and NADPH when bound to different enzymes may therefore be attributed to differing levels of conformational restriction upon binding.

  7. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  8. A COUPLED MICROSOMAL-ACTIVATING/EMBRYO CULTURE SYSTEM: TOXICITY OF REDUCED BETA-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH)

    EPA Science Inventory

    An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decrease in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1....

  9. Lipopolysaccharide (LPS)-mediated angiopoietin-2-dependent autocrine angiogenesis is regulated by NADPH oxidase 2 (Nox2) in human pulmonary microvascular endothelial cells.

    PubMed

    Menden, Heather; Welak, Scott; Cossette, Stephanie; Ramchandran, Ramani; Sampath, Venkatesh

    2015-02-27

    Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of ?-B kinase ? (IKK?) and p38 phosphorylation and nuclear translocation of NF-?B and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKK?/NF-?B and MAPK/AP-1 pathways. PMID:25568324

  10. TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

    SciTech Connect

    St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.; Smith, Barbara D.; Ravid, Katya

    2008-10-24

    Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.

  11. First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis.

    PubMed

    Laleu, Benoît; Gaggini, Francesca; Orchard, Mike; Fioraso-Cartier, Laetitia; Cagnon, Laurène; Houngninou-Molango, Sophie; Gradia, Angelo; Duboux, Guillaume; Merlot, Cédric; Heitz, Freddy; Szyndralewiez, Cédric; Page, Patrick

    2010-11-11

    We describe the design, synthesis, and optimization of first-in-class series of inhibitors of NADPH oxidase isoform 4 (Nox4), an enzyme implicated in several pathologies, in particular idiopathic pulmonary fibrosis, a life-threatening and orphan disease. Initially, several moderately potent pyrazolopyridine dione derivatives were found during a high-throughput screening campaign. SAR investigation around the pyrazolopyridine dione core led to the discovery of several double-digit nanomolar inhibitors in cell free assays of reactive oxygen species (ROS) production, showing high potency on Nox4 and Nox1. The compounds have little affinity for Nox2 isoform and are selective for Nox4/1 isoforms. The specificity of these compounds was confirmed in an extensive in vitro pharmacological profile, as well as in a counterscreening assay for potential ROS scavenging. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo, allowing further clinical trials for the potential treatment of fibrotic diseases, cancers, and cardiovascular and metabolic diseases. PMID:20942471

  12. Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase*

    PubMed Central

    Orru, Roberto; Pazmiño, Daniel E. Torres; Fraaije, Marco W.; Mattevi, Andrea

    2010-01-01

    The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP+ analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this “double-engineering” approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP+ in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications. PMID:20807767

  13. Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

    PubMed Central

    Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

    2013-01-01

    Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

  14. Protective effect of apocynin, a NADPH-oxidase inhibitor, against contrast-induced nephropathy in the diabetic rats: a comparison with n-acetylcysteine.

    PubMed

    Ahmad, Akbar; Mondello, Stefania; Di Paola, Rosanna; Mazzon, Emanuela; Esposito, Emanuela; Catania, Maria Antonietta; Italiano, Domenico; Mondello, Patrizia; Aloisi, Carmela; Cuzzocrea, Salvatore

    2012-01-15

    The aim of this study was to investigate the effects of apocynin, a NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase inhibitor, in diabetic rats with nephropathy induced by contrast medium (CIN). Diabetes was induced in male Wistar rats by a single dose of streptozotocin (60 mg/kg i.v.). Animals were then divided into the following groups: 1) control group (diabetic rats treated i.v. with saline solution); 2) iomeprol group (iomeprol at 10 ml/kg was injected i.v. 30 min after saline administration); 3) apocynin group (identical to the iomeprol group, except for pre-treatment with apocynin 5mg/kg i.v., 30 min before iomeprol injection) and 4) N-acetylcysteine group (NAC) (same as iomeprol group, except for the treatment with NAC 20 mg/kg i.v. 30 min before iomeprol injection). CIN in animals were assessed 24h after administration of iomeprol. Apocynin significantly attenuates the impaired glomerular function, concentration of Na(+), K(+), alpha glutathione S-transferase levels in urine and neutrophil gelatinase-associated lipocalin levels in plasma caused by iomeprol. In kidney, immunohistochemical analysis of some inflammatory mediators, such as nitrotyrosine, poly-ADP-ribosyl polymerase, tumor necrosis factor-?, interleukin-1? as well as apoptosis (evaluated as terminal deoxynucleotidyltransferase-mediated UTP end labeling assay) revealed positive staining in tissue obtained from iomeprol group. These parameters were markedly reduced in animals treated with apocynin. Similarly, these parameters were also markedly modified by NAC pre-treatment. Here, we have shown that apocynin attenuates the degree of iomeprol-induced nephropathy in diabetic rats. PMID:22094062

  15. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  16. Cytosolic Phospholipase A2-Is Necessary for Platelet-activating Factor Biosynthesis, Efficient Neutrophil-mediated Bacterial

    E-print Network

    Gelb, Michael

    Cytosolic Phospholipase A2- Is Necessary for Platelet-activating Factor Biosynthesis, Efficient- ) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation NOT REGULATE NEUTROPHIL NADPH OXIDASE ACTIVITY* Received for publication, July 2, 2004, and in revised form

  17. Association of the NAD(P)H oxidase p22 phox gene C242T polymorphism with type 2 diabetes mellitus, diabetic nephropathy, and carotid atherosclerosis with type 2 diabetes mellitus: A meta-analysis

    PubMed Central

    Li, Tao; Xi, Hai-feng; Luo, Hong-min; Liu, Wen-xuan; Gao, Xia; Liu, Dian-wu; Yang, Lei

    2015-01-01

    Background Several epidemiological studies have evaluated the association between the NAD(P)H oxidase p22 phox gene C242T polymorphism and the risk of type 2 diabetes mellitus (T2DM), diabetic nephropathy (DN), and carotid atherosclerosis with T2DM (CA), but the results are inconclusive. This meta-analysis was therefore designed to clarify these controversies. Methods Systematic searches were performed using electronic databases such as MEDLINE, PubMed, EMBASE, and China National Knowledge Infrastructure, as well as through manual searching of the references of identified articles. A total of 11 publications were eligible for this meta-analysis after running a search on the NAD(P)H oxidase p22 phox gene C242T polymorphism, including 7 with outcomes for T2DM, 7 with outcomes for DN, and 3 with outcomes for CA. The pooled odds ratio (OR) with a 95% confidence interval (CI) was calculated using a fixed effects model (FEM) or a random effects model (REM). Publication bias was tested by Begg's funnel plot analysis. Sensitivity analysis was also performed. Results The results showed a significant association between the NAD(P)H oxidase p22 phox gene C242T polymorphism and T2DM risk in the allelic model (REM: OR = 1.23, 95% CI = 1.06–1.43), additive model (FEM: OR = 1.61, 95% CI = 1.14–2.26), and recessive model (FEM: OR = 1.50, 95% CI = 1.10–2.05). A significant association was also observed for DN in the allelic model (REM: OR = 1.25, 95% CI = 1.06–1.47), additive model (FEM: OR = 1.61, 95% CI = 1.08–2.38), and dominant model (REM: OR = 1.26, 95% CI = 1.03–1.54). However, no association was observed for CA. Similar results were obtained in subgroup analysis based on ethnicity. Conclusions Results of this meta-analysis suggest that the NAD(P)H oxidase p22 phox gene 242T allele might be associated with an increased risk of T2DM and DN, but not CA. PMID:26380814

  18. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    PubMed

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-01

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing. PMID:24174530

  19. Ovarian Dual Oxidase (Duox) Activity Is Essential for Insect Eggshell Hardening and Waterproofing*

    PubMed Central

    Dias, Felipe A.; Gandara, Ana Caroline P.; Queiroz-Barros, Fernanda G.; Oliveira, Raquel L. L.; Sorgine, Marcos H. F.; Braz, Glória R. C.; Oliveira, Pedro L.

    2013-01-01

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing. PMID:24174530

  20. Polyphenol oxidase activity in co-ensiled temperate grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) and its o-diphenol substrates have been shown to effectively decrease proteolytic activity during the ensiling of forages such as red clover. Orchardgrass and smooth bromegrass both contain high levels of PPO activity, but lack appropriate levels of o-diphenols to adequately...

  1. CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

    PubMed Central

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10?6±0.21 M·min?1 and 0.32±0.02 s?1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal. PMID:23577125

  2. Both inducible nitric oxide synthase and NADPH oxidase contribute to the control of virulent phase I Coxiella burnetii infections.

    PubMed

    Brennan, Robert E; Russell, Kasi; Zhang, Guoquan; Samuel, James E

    2004-11-01

    Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-gamma) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-gamma-treated primary macrophages from iNOS-/- and p47phox-/- mice significantly inhibited replication but were less efficient at controlling infection than IFN-gamma-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS-/- and p47phox-/- mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection. PMID:15501800

  3. Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    SciTech Connect

    Flueck, Christa E.; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V.

    2011-09-09

    Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

  4. Effect of mitoguazone on polyamine oxidase activity in rat liver

    SciTech Connect

    Ferioli, Maria Elena . E-mail: MariaElena.Ferioli@unimi.it; Berselli, Debora; Caimi, Samuela

    2004-12-01

    Mitoguazone is a known inhibitor of polyamine biosynthesis through competitive inhibition of S-adenosylmethionine decarboxylase. A recent renewed interest in mitoguazone as an antineoplastic agent prompted us to investigate the effect of the drug on polyamine catabolism in rat liver, since the organ plays an important role in detoxification mechanisms. Thus, the purpose of this work was to evaluate the effect of in vivo mitoguazone administration on polyamine catabolic enzymes. In particular, our interest was directed to the changes in polyamine oxidase activity, since this enzyme has been recently confirmed to exert important functions that until now were underestimated. Mitoguazone administration induced hepatic polyamine oxidase activity starting at 4 h after administration, and the enzyme returned to basal levels 96 h after treatment. The changes in enzyme activity were accompanied by changes in putrescine concentrations, which increased starting at 4 h until 72 h after treatment. We also evaluated the activity of the newly identified spermine oxidase, which was not significantly changed by mitoguazone treatment. Therefore, we hypothesized that the enzyme involved in mitoguazone response of the liver is the polyamine oxidase, which acts on acetylated polyamines as substrate.

  5. Detection of peroxisomal fatty acyl-coenzyme A oxidase activity.

    PubMed Central

    Inestrosa, N C; Bronfman, M; Leighton, F

    1979-01-01

    It has been postulated that the peroxisomal fatty acid-oxidizing system [Lazarow & de Duve (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2043--2046; Lazarow (1978) J. Biol. Chem. 253, 1522--1528] resembles that of mitochondria, except for the first oxidative reaction. In this step, O2 would be directly reduced to H2O2 by an oxidase. Two specific procedures developed to detect the activity of the characteristic enzyme fatty acyl-CoA oxidase are presented, namely polarographic detection of palmitoyl-CoA-dependent cyanide-insensitive O2 consumption and palmitoyl-CoA-dependent H2O2 generation coupled to the peroxidation of methanol in an antimycin A-insensitive reaction. Fatty acyl-CoA oxidase activity is stimulated by FAD, which supports the flavoprotein nature postulated for this enzyme. Its activity increases 7-fold per g wet wt. of liver in rats treated with nafenopin, a hypolipidaemic drug. Subcellular fractionation of livers from normal and nafenopin-treated animals provides evidence for its peroxisomal localization. The stoicheiometry for palmitoyl-CoA-dependent O2 consumption, H2O2 generation and NAD+ reduction is 1 : 1 : 1. This suggests that fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal fatty acid-oxidizing system. PMID:518563

  6. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Sty?a, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Pozna?, West Polish Lowland). The sites of investigation were located along Wysko? ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at ?max=525 nm with catechol as substrate by method of Perucci et al. (2000). In peat the highest activities of phenol oxidase was observed in the combinations marked as Shelterbelt and whereas the lowest - in Zbechy, Bridge and Hirudo. Activities of this enzyme in peat ranged from 15.35 to 38.33 ?mol h-1g d.m soil. Increased activities of phenol oxidase have been recorded on the depth 50-100cm - catotelm (21.74-38.33 ?mol h-1g d.m soil) in comparison with the depth 0-50cm - acrotelm (15.35-28.32 ?mol h-1g d.m soil). References Freeman, C., Ostle N.J., Fener, N., Kang H. 2004. A regulatory role for phenol oxidase during decomposition in peatlands. Soil Biology and Biochemistry, 36, 1663-1667. Matocha Ch.J., Haszler G.R., Grove J.H. 2004. Nitrogen fertilization suppresses soil phenol oxidase enzyme activity in no-tillage systems. Soil Science, 169/10, 708-714. Perucci P., Casucci C., Dumontet S. 2000. An improved method to evaluate the o-diphenol oxidase activity of soil. Soil Biology and Biochemistry, 32, 1927-1933. Sokolowska Z., Szajdak L., Matyka-Sarzy?ska D. 2005. Impact of the degree of secondary transformation on amid-base properties of organic compounds in mucks. Geoderma, 127, 80-90. Szajdak L., Szczepa?ski M., Bogacz A. 2007. Impact of secondary transformation of peat-moorsh soils on the decrease of nitrogen and carbon compounds in ground water. Agronomy Research, 5/2, 189-200.

  7. Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

    PubMed

    Palmieri, Erika M; Spera, Iolanda; Menga, Alessio; Infantino, Vittoria; Porcelli, Vito; Iacobazzi, Vito; Pierri, Ciro L; Hooper, Douglas C; Palmieri, Ferdinando; Castegna, Alessandra

    2015-08-01

    The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme. PMID:25917893

  8. The nitrate reductase activity of milk xanthine oxidase.

    PubMed

    Sergeev, N S; Ananiadi, L I; L'vov, N P; Kretovich, W L

    1985-04-01

    Milk xanthine oxidase oxidizes xanthine at pH 9.6 and reduces nitrates at pH 5.2. It is shown that the nitrate reductase activity requires molybdenum and sulfur-containing sites in the enzyme, whereas oxidation of xanthine also requires iron-containing sites and FAD. As the pH changes from 5.2 to 9.6, the conformation of the enzyme molecule is modified as demonstrated by changes in the absorption, fluorescence, and circular dichroism spectra. When the enzyme is treated with dithioerythritol, it may pass from the oxidase to the dehydrogenase form with a marked increase in the nitrate reductase activity. PMID:3840469

  9. Potential xanthine oxidase inhibitory activity of endophytic Lasiodiplodia pseudotheobromae.

    PubMed

    Kapoor, Neha; Saxena, Sanjai

    2014-07-01

    Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC50 value of 0.61 ?g ml(-1) which was better than allopurinol exhibiting an IC50 of 0.937 ?g ml(-1) while febuxostat exhibited a much lower IC50 of 0.076 ?g ml(-1). Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity. PMID:24801403

  10. Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

    PubMed Central

    Kim, Sook-Hee; Choi, Haymie

    2008-01-01

    Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth. PMID:20126370

  11. Cofactor Specificity Engineering of Streptococcus mutans NADH Oxidase 2 for NAD(P)+ Regeneration in Biocatalytic Oxidations

    PubMed Central

    Petschacher, Barbara; Staunig, Nicole; Müller, Monika; Schürmann, Martin; Mink, Daniel; De Wildeman, Stefaan; Gruber, Karl; Glieder, Anton

    2014-01-01

    Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)+ regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 193R194H showed comparable high rates and low K m values for NADPH (k cat 20 s-1, K m 6 µM) and NADH (k cat 25 s-1, K m 9 µM) with retention of 70% of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production. PMID:24757503

  12. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies

    PubMed Central

    Andjelkovi?, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T.

    2015-01-01

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. PMID:26672986

  13. Increased activity of monoamine oxidase by epoxy resin hardeners.

    PubMed

    Yano, E

    1987-06-01

    In order to investigate the potency of amines to cause metabolic changes which are related to scleroderma, serum monoamine oxidase (MAO) activity to workers in an epoxy resin handling process was measured. Mean serum MAO activity of 15 workers exposed to amine was 33.5 +/- 6.4 units, whilst that of control workers was 28.9 +/- 7.8 (P less than 0.05). The finding was confirmed in an in vitro experiment. After 24 h treatment with amine, the MAO activity of cultured skin fibroblasts was elevated in a dose-response manner. These results suggested the potency of amine to produce an increase in MAO activity, and this phenomenon seems to be related to the cases of occupational scleroderma-like disorder which were observed in an epoxy resin polymerizing process. PMID:3590227

  14. [Experimental rationale for the parameters of a rapid method for oxidase activity determination].

    PubMed

    Butorina, N N

    2010-01-01

    Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005. PMID:21341494

  15. [Functional groups involved in the nitrate reductase activity of milk xanthine oxidase].

    PubMed

    Ananiadi, L I; Sergeev, N S; Kil'dibekov, N A; L'vov, N P; Kretovich, V L

    1983-06-01

    Milk xanthine oxidase possesses the nitrate reductase activity at pH 5.2; the pH optimum of the xanthine oxidase activity for the enzyme lies at 9.6. After removal of FAD and binding of Mo and Fe with a simultaneous measurement at the pH optima of the above activities it was found that only the Mo-containing site is necessary for the nitrate reductase activity. The switch-over of the enzyme from the xanthine oxidase to the nitrate reductase activity is associated with considerable conformational changes of the enzyme molecule. PMID:6688366

  16. Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host-pathogen interaction.

    PubMed

    Schürmann, Janine; Buttermann, Dagmar; Herrmann, Andrea; Giesbert, Sabine; Tudzynski, Paul

    2013-10-01

    Reactive oxygen species producing NADPH oxidase (Nox) complexes are involved in defense reactions in animals and plants while they trigger infection-related processes in pathogenic fungi. Knowledge about the composition and localization of these complexes in fungi is limited; potential components identified thus far include two to three catalytical subunits, a regulatory subunit (NoxR), the GTPase Rac, the scaffold protein Bem1, and a tetraspanin-like membrane protein (Pls1). We showed that, in the biotrophic grass-pathogen Claviceps purpurea, the catalytical subunit CpNox1 is important for infection. Here, we present identification of major Nox complex partners and a functional analysis of CpNox2 and the tetraspanin CpPls1. We show that, as in other fungi, Nox complexes are important for formation of sclerotia; CpRac is, indeed, a complex partner because it interacts with CpNoxR, and CpNox1/2 and CpPls1 are associated with the endoplasmatic reticulum. However, unlike in all other fungi, ?cppls1 is more similar to ?cpnox1 than to ?cpnox2, and CpNox2 is not essential for infection. In contrast, ?cpnox2 shows even more pronounced disease symptoms, indicating that Cpnox2 controls the infection process and moderates damage to the host. These data confirm that fungal Nox complexes have acquired specific functions dependent of the lifestyle of the pathogen. PMID:23777432

  17. A P2X7 receptor antagonist attenuates experimental autoimmune myocarditis via suppressed myocardial CD4+ T and macrophage infiltration and NADPH oxidase 2/4 expression in mice.

    PubMed

    Zempo, Hirofumi; Sugita, Yoichiro; Ogawa, Masahito; Watanabe, Ryo; Suzuki, Jun-Ichi; Isobe, Mitsuaki

    2015-07-01

    Myocarditis is a clinically serious disease; however, no effective treatment has been elucidated. The P2X7 receptor is related to the pathophysiology of inflammation in many cardiovascular diseases. The P2X7 receptor antagonist is promising as an immunosuppressive treatment; however, its role in myocarditis is still to be established. To clarify the role of the P2X7 receptor, we used a murine experimental autoimmune myocarditis (EAM) model. Mice were immunized on day 0 and 7 with synthetic cardiac myosin peptide to establish EAM. The mice with induced EAM were treated with A740003, the P2X7 receptor antagonist (n = 10), or not treated (n = 11); hearts were harvested on day 21. The P2X7 receptor antagonist improved myocardial contraction of the EAM hearts via suppressed infiltration of CD4+ T cells and macrophages. Similarly, mRNA expression of interleukin 1 beta, the P2X7 receptor and NADPH oxidase 2/4 was lower in the heart of the P2X7 receptor antagonist-treated group compared to the non-treat group. The P2X7 receptor antagonist suppressed EAM development; thus, this inhibition is promising for treating clinical myocarditis. PMID:24879505

  18. Inhibition of apple polyphenol oxidase activity by sodium chlorite.

    PubMed

    Lu, Shengmin; Luo, Yaguang; Feng, Hao

    2006-05-17

    Sodium chlorite (SC) was shown to have strong efficacy both as a sanitizer to reduce microbial growth on produce and as a browning inhibitor on fresh-cut apples in previous experiments. This study was undertaken to investigate the inhibitory effect of SC on polyphenol oxidase (PPO) and the associated mechanisms. The experiment showed that SC had a strong inhibition of apple PPO. The extent of inhibition was influenced by SC concentration and pH. Inhibition was most prominent at pH 4.5, at which approximately 30% of enzyme activity was lost in the presence of 10 mM SC, followed closely by that at pH 4.0 with a 26% reduction in PPO activity. The inhibition mode was determined using Dixon and Lineweaver-Burk plots, which established SC to be a mixed inhibitor of apple PPO for the oxidation of catechol. Preincubation of PPO with 8 mM SC for 8 min caused a maximum of 46% activity reduction compared to noninhibited control. However, preincubation of SC with catechol for 8 min resulted in no additional loss of PPO activity. These findings provide further evidence that the inhibition of PPO activity by SC is due to the inhibition of the enzyme itself rather than removal of the substrate. PMID:19127746

  19. Inhibition and oxygen activation in copper amine oxidases.

    PubMed

    Shepard, Eric M; Dooley, David M

    2015-05-19

    Copper-containing amine oxidases (CuAOs) use both copper and 2,4,5-trihydroxyphenylalanine quinone (TPQ) to catalyze the oxidative deamination of primary amines. The CuAO active site is highly conserved and comprised of TPQ and a mononuclear type II copper center that exhibits five-coordinate, distorted square pyramidal coordination geometry with histidine ligands and equatorially and axially bound water in the oxidized, resting state. The active site is buried within the protein, and CuAOs from various sources display remarkable diversity with respect to the composition of the active site channel and cofactor accessibility. Structural and mechanistic factors that influence substrate preference and inhibitor sensitivity and selectivity have been defined. This Account summarizes the strategies used to design selective CuAO inhibitors based on active site channel characteristics, leading to either enhanced steric fits or the trapping of reactive electrophilic products. These findings provide a framework to support the future development of candidate molecules aimed at minimizing the negative side effects associated with drugs containing amine functionalities. This is vital given the existence of human diamine oxidase and vascular adhesion protein-1, which have distinct amine substrate preferences and are associated with different metabolic processes. Inhibition of these enzymes by antifungal or antiprotozoal agents, as well as classic monoamine oxidase (MAO) inhibitors, may contribute to the adverse side effects associated with drug treatment. These observations provide a rationale for the limited clinical value associated with certain amine-containing pharmaceuticals and emphasize the need for more selective AO inhibitors. This Account also discusses the novel roles of copper and TPQ in the chemistry of O2 activation and substrate oxidation. Reduced CuAOs exist in a redox equilibrium between the Cu(II)-TPQAMQ (aminoquinol) and Cu(I)-TPQSQ (semiquinone). Elucidating the roles of Cu(I), TPQSQ, and TPQAMQ in O2 activation, for example, distinguishing inner-sphere versus outer-sphere electron transfer mechanisms, has been actively investigated since the discovery of TPQSQ in 1991 and has only recently been clarified. Kinetics and spectroscopic studies encompassing metal substitution, stopped-flow and temperature-jump relaxation methods, and oxygen kinetic isotope experiments have provided strong support for an inner-sphere electron transfer step from Cu(I) to O2. Data for two enzymes support a mechanism wherein O2 prebinds to a three-coordinate Cu(I) site, yielding a [Cu(II)(?(1)-O2(-1))](+) intermediate, with H2O2 generated from ensuing rate-determining proton coupled electron transfer from TPQSQ. While kinetics data from the cobalt-substituted yeast enzyme indicated that O2 is reduced through an outer-sphere process involving TPQAMQ, new findings with a bacterial CuAO demonstrate that both the Cu(II) and Co(II) forms of the enzyme operate via parallel mechanisms involving metal-superoxide intermediates. Structural observations of a coordinated TPQSQ-Cu(I) complex in two CuAOs supports previous indications that Cu(II)/(I) ligand substitution chemistry may be mechanistically relevant. Substantial evidence indicates that rapid and reversible inner-sphere reduction of O2 at a three-coordinate Cu(I) site occurs, but the existence of a coordinated semiquinone in some AOs suggests that, in these enzymes, an outer-sphere reaction between O2 and TPQSQ may also be possible, since this is expected to be energetically favorable compared with outer-sphere electron transfer from TPQAMQ to O2. PMID:25897668

  20. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa. PMID:25308684

  1. Xanthine oxidase inhibitory activity of extracts prepared from Polygonaceae species.

    PubMed

    Orbán-Gyapai, Orsolya; Lajter, Ildikó; Hohmann, Judit; Jakab, Gusztáv; Vasas, Andrea

    2015-03-01

    The xanthine oxidase (XO) inhibitory activity of aqueous and organic extracts of 27 selected species belonging in five genera (Fallopia, Oxyria, Persicaria, Polygonum and Rumex) of the family Polygonaceae occurring in the Carpathian Basin were tested in vitro. From different plant parts (aerial parts, leaves, flowers, fruits and roots), a total of 196 extracts were prepared by subsequent extraction with methanol and hot H2O and solvent-solvent partition of the MeOH extract yielding n-hexane, chloroform and 50% MeOH subextracts. It was found that the chloroform subextracts and/or the remaining 50% MeOH extracts of Fallopia species (F. bohemica, F. japonica and F. sachalinensis), Rumex species (R. acetosa, R. acetosella, R. alpinus, R. conglomeratus, R. crispus, R. hydrolapathus, R. pulcher, R. stenophyllus, R. thyrsiflorus, R. obtusifolius subsp. subalpinus, R. patientia) and Polygonum bistorta, Polygonum hydropiper, Polygonum lapathifolium and Polygonum viviparum demonstrated the highest XO inhibitory activity (>85% inhibition) at 400?µg/mL. The IC50 values of the active extracts were also determined. On the basis of the results, these plants, and especially P. hydropiper and R. acetosella, are considered worthy of activity-guided phytochemical investigations. PMID:25510560

  2. Channelling and formation of ‘active’ formaldehyde in dimethylglycine oxidase

    PubMed Central

    Leys, David; Basran, Jaswir; Scrutton, Nigel S.

    2003-01-01

    Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are ?40 ? apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry–exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms. PMID:12912903

  3. Unfolding and refolding of active apple polyphenol oxidase.

    PubMed

    Mari, S; Marquès, L; Breton, F; Karamanos, Y; Macheix, J J

    1998-11-01

    For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases. PMID:9842726

  4. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  5. Effect of zinc deficiency on NADPH and cytochrome P-450 dependent active oxygen generation in rat lung and liver

    SciTech Connect

    Hammermueller, J.D.; Bray, T.M.; Bettger, W.J.

    1986-03-05

    The cyt. P-450 system and cyt. P-450 reductase are involved in the generation of active oxygen species such as H/sub 2/O/sub 2/. The objective of this study was to investigate the effect of short term, severe, dietary zinc deficiency in rats on the formation of active oxygen in vitro. Weanling male Wistar rats were fed egg white-based diets containing less than 1 ppm Zn (ZnD). Controls were fed ad libitum (ZnAl) or pair-fed (ZnPF) a diet containing 100 ppm Zn. After 3 weeks lung and liver microsomes were assayed for H/sub 2/O/sub 2/ production (pmol H/sub 2/O/sub 2//mg protein/min) and cyt. P-450 reductase activity (nmol cyt. C reduced/mg protein/min). For the measurement of H/sub 2/O/sub 2/ production exogenous substrate (aminopyrine) and NADPH (cofactor) were provided to drive the cyt. P-450 system and NaN/sub 3/ was used to inhibit catalase. The results showed a significant effect of dietary Zn on NADPH and cyt. P-450 dependent active oxygen generation and support the hypothesis that Zn has a role in the function of biomembranes.

  6. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%. PMID:15137808

  7. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  8. ATP modulates acute inflammation in vivo through dual oxidase 1-derived H2O2 production and NF-?B activation.

    PubMed

    de Oliveira, Sofia; López-Muñoz, Azucena; Candel, Sergio; Pelegrín, Pablo; Calado, Ângelo; Mulero, Victoriano

    2014-06-15

    Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H2O2 gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H2O2 release from DUOX1 and whether the H2O2 gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H2O2 release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H2O2 is able to trigger the NF-?B inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca(2+) signaling pathway and the production of H2O2, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-?B signaling pathway. PMID:24842759

  9. Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity1[C][W

    PubMed Central

    Kim, Dong Wook; Watanabe, Kanako; Murayama, Chihiro; Izawa, Sho; Niitsu, Masaru; Michael, Anthony J.; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development. PMID:24906355

  10. Ascorbic acid potentiates the substrate-specific inhibition of mixed-function oxidation and the stimulation of NADPH oxidation caused by paraquat.

    PubMed

    Montgomery, M R; Shamblin, P B

    1984-01-01

    Paraquat inhibits the in vitro hepatic microsomal metabolism of both ethylmorphine and aniline. Inclusion of ascorbate with paraquat in the incubations did not alter the paraquat effect on ethylmorphine N-demethylase activity but potentiated the inhibition of aniline p-hydroxylase activity. Ascorbate alone was without effect on the metabolism of either substrate. Paraquat stimulated the hepatic microsomal oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) equally in the absence of mixed-function oxidase (MFO) substrates or in the presence of ethylmorphine; in the presence of aniline the rate of NADPH oxidation was significantly greater. Also, in the presence of aniline, ascorbate potentiated the paraquat-induced NADPH oxidation, while it was ineffective with paraquat on NADPH oxidation in the presence of ethylmorphine or in the absence of substrates for the microsomal MFO system. The potentiated inhibition of aniline metabolism, concomitant with the potentiated stimulation of NADPH oxidation, was consistent whether liver microsomal fractions were prepared from control rats or from animals induced with phenobarbital. Investigation of possible influences on NADPH cytochrome c reductase activity was precluded by the rapid nonenzymatic reduction of cytochrome c by ascorbate. The paraquat-ascorbate redox couple would not reduce cytochrome P-450. These data suggest that a paraquat interaction with the active microsomal MFO enzyme system plays a role in the depletion of cellular NADPH stores that occurs after paraquat administration in vivo. This mechanism may play a significant role in the development of paraquat toxicity and in the potentiated toxicity observed with ascorbate and paraquat. PMID:6716512

  11. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6?-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3?-hydroxysteroids such as cholesterol, ?-cholestanol, and ?-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 ?M and 15.9 ?mol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa. PMID:25573142

  12. Bifunctionalized mesoporous silica-supported gold nanoparticles: intrinsic oxidase and peroxidase catalytic activities for antibacterial applications.

    PubMed

    Tao, Yu; Ju, Enguo; Ren, Jinsong; Qu, Xiaogang

    2015-02-11

    Bifunctionalized mesoporous silica-supported gold nanoparticles as oxidase and peroxidase mimics for antibacterial applications are demonstrated. For the first time, these mesoporous silica-supported gold nanoparticles are applied as oxidase and peroxidase mimics. Taking advantage of their prominent enzyme activities, the MSN-AuNPs show excellent antibacterial properties against both Gram-negative and Gram-positive bacteria. Furthermore, MSN-AuNPs also exhibit outstanding performance in biofilm elimination . PMID:25655182

  13. Genetic control of aldehyde oxidase activity and cross-reacting-material in Drosophila melanogaster.

    PubMed

    Meidinger, E M; Williamson, J H

    1978-12-01

    Four different genes are known to affect aldehyde oxidase activity (AO) in Drosophila melanogaster. Mutants at each of these loci eliminate AO activity and simultaneously eliminate detectable AO-crossing reacting material (AO-CRM) even though only one is the structural gene for AO (Aldoxn). The other three genes (cin1, lxd and mal) coordinately "control" the levels of activity of AO and two related enzymes, xanthine dehydrogenase (XDH) and pyridoxal oxidase (PO). Contrary to their effects on AO-CRM, neither of these three mutants eliminate XDH-CRM. A model of interaction of these enzymes and genes controlling their activities is discussed. PMID:94842

  14. Transforming Growth Factor ?1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis.

    PubMed

    Das, Ranjan; Xu, Shanhua; Nguyen, Tuyet Thi; Quan, Xianglan; Choi, Seong-Kyung; Kim, Soo-Jin; Lee, Eun Young; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-12-25

    TGF-? is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-?1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-?1-induced apoptosis in immortalized podocytes. TGF-?1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-? receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTOR? augmented TGF-?1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-?1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-?1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-?1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-?1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-?1-induced ERK1/2-mTORactivation. Our data suggest that TGF-?1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases. PMID:26565025

  15. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Ye?iller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. PMID:26257292

  16. Excess boron reduces polyphenol oxidase activities in embryo and endosperm of maize seed during germination.

    PubMed

    Olçer, Hillya; Kocaçaliskan, Ismail

    2007-01-01

    The effects of increasing concentrations of boron (0, 0.1, 1, 10 and 20 mM) as boric acid on the rate of germination and polyphenol oxidase activities in embryo and endosperm tissues of maize seeds (Zea mays L. cv. Arifiye) were studied. The germination percentage of maize seeds was not affected by boron concentrations up to 10 mM, and decreased by 20 mM. Distilled water and lower boron concentrations (0.1 and 1 mM) increased polyphenol oxidase activities at the beginning of germination up to 12 h whereas its excess levels (10 and 20 mM) decreased polyphenol oxidase activities in embryos and endosperm during germination. Polyphenol oxidase activities with o-diphenolic substrates (caffeic acid, catechol and dopa) were found to be higher than with a monophenolic substrat (tyrosine) in both embryos and endosperms. Further, caffeic acid oxidizing polyphenol oxidase was found to show more activity in embryos of the seeds germinating in distilled water when compared to other substrates. PMID:17425115

  17. Ginkgolide B protects against cisplatin-induced ototoxicity: enhancement of Akt-Nrf2-HO-1 signaling and reduction of NADPH oxidase.

    PubMed

    Ma, Weijun; Hu, Juan; Cheng, Ying; Wang, Junli; Zhang, Xiaotong; Xu, Min

    2015-05-01

    Cisplatin is a widely used chemotherapeutic drug for the treatment of various cancers. However, the ototoxicity severely limited its maximum dose. The present study was designed to evaluate the effect of Ginkgolide B (GB), a major component of Ginkgo biloba extracts, on cisplatin-induced ototoxicity and to elucidate the molecular mechanism in vitro and in vivo. In HEI-OC1 auditory cells, GB concentration-dependently inhibited the reduction of cell viability and increase in apoptosis exerted by cisplatin. Cisplatin-activated mitochondrial apoptotic molecular events were significantly inhibited by GB. In addition, GB notably suppressed the increase in NOX2 and p47(phox) expression and the decrease in nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in cisplatin-exposed cells. Inhibition of Nrf2 using SiRNA and blockage of HO-1 by zinc protoporphyrin IX (ZnPP) suppressed the protective effects of GB. Moreover, GB prevented cisplatin-induced reduction of Akt phosphorylation and LY294002, an inhibitor of PI3 K/Akt signaling, blocked the anti-apoptotic effect of GB in cisplatin-treated cells. Furthermore, the protective effect of GB was tested in cisplatin-exposed rats. GB treatment markedly protected animals against cisplatin-induced hearing loss and vestibular dysfunction. Inhibition of Akt and HO-1 significantly suppressed the improvement in hearing loss and vestibular dysfunction in GB-treated rats. We demonstrate that GB decreases ROS generation through reducing NOX2 expression and enhancing activity through Akt-Nrf2-HO-1 pathway, resulting in inhibition of mitochondrial apoptosis and final reduction of cisplatin-induced ototoxicity in vitro and in vivo. Our findings have gained an insight into the mechanism of GB-exerted protective effect against cisplatin-induced ototoxicity. PMID:25749575

  18. Diphenol activation of the monophenolase and diphenolase activities of field bean (Dolichos lablab) polyphenol oxidase.

    PubMed

    Gowda, Lalitha R; Paul, Beena

    2002-03-13

    This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean (Dolichos lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which decreased with increasing catechol concentration and increased with increasing ferulic acid concentration. The activation constant K(a) of catechol for ferulic acid hydroxylation reaction was 5 mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and 2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase, could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase (Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine, and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs immediately with no lag period and does not occur without the addition of diphenol. The kinetic parameters for caffeic acid (K(m) = 0.08 mM, V(max) = 32440 u/mg) in the presence of catechol and the activation constant K(a) of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag period for this reaction indicates that the diphenol mechanism of diphenolase activation differs from the way in which the same o-diphenols activate the monophenolase activity. PMID:11879044

  19. 1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

    PubMed Central

    Vriezen, Wim H.; Hulzink, Raymond; Mariani, Celestina; Voesenek, Laurentius A.C.J.

    1999-01-01

    Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase in RP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783–791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustris during submergence. PMID:10482674

  20. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  1. Inheritance of grain polyphenol oxidase (PPO) activity in multiple wheat (Triticum aestivum L.) genetic backgrounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS marker...

  2. A NOVEL STS MARKER FOR POLYPHENOL OXIDASE ACTIVITY IN BREAD WHEAT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme activity of polyphenol oxidase (PPO) in grain has been related to undersirable brown discoloration of bread wheat (Triticum aestivum L.) based end-products, particularly for Asian noodles. Breeding wheat cultivars with low PPO activity is the best approach to restrain reduce the undesirab...

  3. Virtual Screening Analysis and In-vitro Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids

    PubMed Central

    Umamaheswari, Muthuswamy; Madeswaran, Arumugam; Asokkumar, Kuppusamy

    2013-01-01

    Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of gout. We examined the xanthine oxidase inhibitory activity of some commercially available flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by virtual screening and in-vitro studies. The interacting residues within the complex model and their contact types were identified. The virtual screening analysis were carried out using AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis. Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/ mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/ mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of xanthine oxidase due to their active binding sites and lesser binding energies compared to allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 ?g/mL, whereas that of allopurinol was 24±0.28 ?g/mL. All the remaining compounds exhibited IC50 values ranging between 22±0.64 to 62±1.18 ?g/mL. In the enzyme kinetic studies, flavonoids showed competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are required to develop potential compounds with lesser side effects. PMID:24250638

  4. Two peanut germin-like genes and the potential superoxidase dismutase and oxalate oxidase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Germins and germin-like proteins (GLPs) genes are members of large multigene families. These genes have been reported to play a role directly or indirectly in plant defense response. A number of GLPs have been demonstrated to have superoxidase dismutase (SOD) or oxalate oxidase (OxO) activity leadin...

  5. POLYPHENOL OXIDASE IN WHEAT GRAIN: WHOLE KERNEL AND BRAN ASSAYS FOR TOTAL AND SOLUBLE ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Color is a key quality trait of wheat products, and polyphenol oxidase (PPO) is implicated as playing a significant role in their darkening and discoloration. In this study, total and soluble PPO activities were characterized in whole-kernel assays and bran extracts. In whole-kernel assays similar...

  6. CLONING OF RED CLOVER POLYPHENOL OXIDASE CDNAS AND CHARACTERIZATION OF ACTIVE PROTEIN EXPRESSED IN TRANSGENIC ALFALFA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover contains high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates resulting in a characteristic post-harvest browning reaction associated with decreased protein degradation during ensiling. To define PPO's role in inhibiting post-harvest proteolysis, we are using both bi...

  7. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    SciTech Connect

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  8. INVESTIGATIONS OF AMITRAZ NEUROTOXICITY IN RATS. III. EFFECTS ON MOTOR ACTIVITY AND INHIBITION OF MONOAMINE OXIDASE

    EPA Science Inventory

    The formamidine pesticide amitraz (AMZ) produces many behavioral and physiological changes in rats. o explore possible neurochemical mechanisms for the behavioral effects of AMZ, we examined the dose effect and time course of AMZ on motor activity, monoamine oxidase (MAO) and ace...

  9. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  10. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed. PMID:11451442

  11. Artificial Warming and Rain Addition Increase Phenol Oxidase Activity in Arctic Soils

    NASA Astrophysics Data System (ADS)

    Kang, H.; Seo, J.; Jang, I.; Lee, Y. K.

    2014-12-01

    Artic tundra is one of the largest carbon stocks, of which amount is estimated up to 1,600 Pg. Global climate change models predict surface temperature rise and higher precipitation during summer in Arctic regions, raising concerns about faster decomposition of organic carbon and consequent releases of CO2, CH4 and DOC. Microorganisms are directly involved in decomposition process by releasing various extracellular enzymes. In particular, phenol oxidase was noted to play a key role because it is related to dynamics of highly recalcitrant carbon, which often represents a rate-limiting step of overall decomposition. In this study, we monitored phenol oxidase activity, hydrolases (?-glucosidase, cellobiohydrolase, N-acetylglucosaminidase and aminopeptidase), microbial abundance (qPCR) and chemical properties (?13C and ?15N signatures) of tundra soils exposed to artificial warming and rain addition, by employing a passive chamber method in Cambridge Bay, Canada. Warming and rain addition combinedly increased phenol oxidase activity while no such changes were discernible for other hydrolases. Stable isotope signature indicates that warming induced water stress to the ecosystem and that nitrogen availability may be enhanced, which is partially responsible for the changes in enzyme activities. A short-term warming (2 years) may not accelerate mineralization of easily decomposable carbon, but may affect phenol oxidase which has the longer-term influence on recalcitrant carbon.

  12. Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

    PubMed Central

    Mercier, N; Moldes, M; El Hadri, K; Fève, B

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

  13. Molecular Basis of Reduced Pyridoxine 5?-Phosphate Oxidase Catalytic Activity in Neonatal Epileptic Encephalopathy Disorder*

    PubMed Central

    Musayev, Faik N.; Di Salvo, Martino L.; Saavedra, Mario A.; Contestabile, Roberto; Ghatge, Mohini S.; Haynes, Alexina; Schirch, Verne; Safo, Martin K.

    2009-01-01

    Mutations in pyridoxine 5?-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5?-phosphate oxidase catalyzes the oxidation of pyridoxine 5?-phosphate to pyridoxal 5?-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ?850-fold less efficient than the wild-type enzyme due to an ?192-fold decrease in pyridoxine 5?-phosphate affinity and an ?4.5-fold decrease in catalytic activity. There is also an ?50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a ?-strand-loop-?-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5?-phosphate oxidase mutations. PMID:19759001

  14. Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin.

    PubMed

    Lennon, A M

    1992-11-15

    A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin. PMID:1446686

  15. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  16. Structure-based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction†

    PubMed Central

    Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

    2012-01-01

    Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate Reductase is an evolutionally related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. Based on sequence alignments and the known crystal structure of chicken sulfite oxidase two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. Based on the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore the triple variant was also produced. The nitrate-reducing ability of the human sulfite oxidase triple mutant was nearly three-fold higher than that of the double mutant. In order to obtain detailed structural data on the active site of these variants, the analogous mutations in chicken sulfite oxidase were generated in order to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were solved to 2.4 Å and 2.1Å resolution, respectively. PMID:22263579

  17. Activity of carbohydrate oxidases as influenced by wheat flour dough components.

    PubMed

    Degrand, L; Rakotozafy, L; Nicolas, J

    2015-08-15

    The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-?-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation. PMID:25794758

  18. Aldehyde oxidases of Drosophila: contributions of several enzymes to observed activity patterns.

    PubMed

    Dickinson, W J; Gaughan, S

    1981-06-01

    At least four enzymes contribute to histochemically, electrophoretically, or spectrophotometrically detectable aldehyde oxidase (AO) activity in Drosophila melanogaster. The one we designate AO-1 contributes the majority of activity measured in extracts of whole flies. Pyridoxal oxidase (PO) is also a broad range AO. It is prominent only in midgut and Malpighian tubules, where it apparently accounts for a substantial fraction of total AO activity. The tissue distributions of these enzymes are clearly disparate despite close linkage of their structural loci and parallel dependence on the mal, lxd, and cin loci. A similarly related enzyme, xanthine dehydrogenase (XDH), is detected as an AO only in electrophoretic gels. A fourth broad range AO, not dependent on mal, lxd, and cin, is confined to the ejaculatory bulb. A similar array of AO isozymes is present in phylogenetically distant Drosophila species. PMID:6794563

  19. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

  20. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    PubMed Central

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  1. 4-Hydroxyanisole: the most suitable monophenolic substrate for determining spectrophotometrically the monophenolase activity of polyphenol oxidase from fruits and vegetables.

    PubMed

    Espín, J C; Tudela, J; García-Cánovas, F

    1998-05-15

    A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those of p-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolas activity of polyphenol oxidase from fruits and vegetables. PMID:9606152

  2. Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

    PubMed Central

    Sinigaglia, Giulietta; Magro, Massimiliano; Miotto, Giovanni; Cardillo, Sara; Agostinelli, Enzo; Zboril, Radek; Bidollari, Eris; Vianello, Fabio

    2012-01-01

    Novel superparamagnetic surface-active maghemite nanoparticles (SAMNs) characterized by a diameter of 10 ± 2 nm were modified with bovine serum amine oxidase, which used rhodamine B isothiocyanate (RITC) adduct as a fluorescent spacer-arm. A fluorescent and magnetically drivable adduct comprised of bovine serum copper-containing amine oxidase (SAMN–RITC–BSAO) that immobilized on the surface of specifically functionalized magnetic nanoparticles was developed. The multifunctional nanomaterial was characterized using transmission electron microscopy, infrared spectroscopy, mass spectrometry, and activity measurements. The results of this study demonstrated that bare magnetic nanoparticles form stable colloidal suspensions in aqueous solutions. The maximum binding capacity of bovine serum amine oxidase was approximately 6.4 mg g?1 nanoparticles. The immobilization procedure reduced the catalytic activity of the native enzyme to 30% ± 10% and the Michaelis constant was increased by a factor of 2. We suggest that the SAMN–RITC–BSAO complex, characterized by a specific activity of 0.81 IU g?1, could be used in the presence of polyamines to create a fluorescent magnetically drivable H2O2 and aldehydes-producing system. Selective tumor cell destruction is suggested as a potential future application of this system. PMID:22619559

  3. Reduced cytochrome oxidase activity in the retrosplenial cortex after lesions to the anterior thalamic nuclei.

    PubMed

    Mendez-Lopez, Magdalena; Arias, Jorge L; Bontempi, Bruno; Wolff, Mathieu

    2013-08-01

    The anterior thalamic nuclei (ATN) make a critical contribution to hippocampal system functions. Growing experimental work shows that the effects of ATN lesions often resemble those of hippocampal lesions and both markedly reduce the expression of immediate-early gene markers in the retrosplenial cortex, which still appears normal by standard histological means. This study shows that moderate ATN damage was sufficient to produce severe spatial memory impairment as measured in a radial-arm maze. Furthermore, ATN rats exhibited reduced cytochrome oxidase activity in the most superficial cortical layers of the granular retrosplenial cortex, and, to a lesser extent, in the anterior cingulate cortex. By contrast, no change in cytochrome oxidase activity was observed in other limbic cortical regions or in the hippocampal formation. Altogether our results indicate that endogenous long-term brain metabolic capacity within the granular retrosplenial cortex is compromised by even limited ATN damage. PMID:23660649

  4. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  5. Effects of dietary carbohydrate on iron metabolism and cytochrome oxidase activity in copper-deficient rats

    SciTech Connect

    Johnson, M.A.; Henderson, J.

    1986-03-01

    The effects of dietary carbohydrate on the metabolism of iron and the activity of cytochrome oxidase were examined in Cu-deficient and Cu-adequate rats. Male rats (n = 36) were fed one of six diets which varied in copper level (Cu-: < 0.6 ppm or Cu+: 8.2 ppm) and carbohydrate type (cornstarch, sucrose or fructose). After 31 days, Cu- rats had 50% more iron in the liver and 38, 30 and 18% less iron in the tibia, spleen and kidneys, respectively, than Cu+ rats. The activity of cytochrome oxidase in the bone marrow, heart, and liver were 59%, 51%, and 43%, respectively, of the levels in Cu/sup +/ rats. The type of dietary carbohydrate significantly affected the development of anemia during copper deficiency. Cu-rats fed cornstarch, sucrose or fructose had hematocrit levels which were 92, 83 or 73%, respectively, of Cu+ rats. Similarly, the levels of iron in the tibias of Cu- rats fed cornstarch, sucrose or fructose were 69, 66 or 54%, respectively, of Cu+ rats. The hematocrit levels of Cu- rats were positively correlated to both tibia iron levels (r = 0.64, p < 0.005) and liver cytochrome oxidase activities (r = 0.50, p < 0.05). Thus, it appears that changes in the metabolism of iron may be involved with the development of anemia in Cu- rats fed fructose or sucrose.

  6. Activation of tobacco leaf polyphenol oxidase by sodium dodecyl sulfate.

    PubMed

    Jiang, Hui; Shi, Chunhua; Xie, Yongshu; Xu, Xiaolong; Liu, Qingliang

    2003-10-01

    The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10 degrees C, suggesting that stabilization of the structure had been improved by the formation of complex. PMID:22900329

  7. Enhanced hydrolysis of soluble cellulosic substrates by a metallocellulase with veratryl alcohol-oxidase activity

    SciTech Connect

    Evans, B.R.; Margalt, R.; Woodward, J.

    1995-12-31

    A cellulose enzyme fraction was separated from Trichoderma reesei Pulpzyme HA{trademark}, and its characteristics suggested that it was mainly composed of cellobiohydrolase II (CBH II). The covalent attachment of pentaammineruthenium (III) to this enzyme resulted in threefold and fourfold enhancements of its hydrolytic activity on carboxymethyl cellulose (CMC) and barley {beta}-glucan, respectively, as well as endowing it with veratryl alcohol-oxidase activity. Enhancement of hydrolysis was not affected by addition of tartrate or hydrogen peroxide to the reaction mixture. Both native and pentaammineruthenium modified enzymes had negligible activity on cellobiose and p-nitrophenyl {beta}-cellobioside (PNPC).

  8. Effect of architecture on the activity of glucose oxidase/horseradish peroxidase/carbon nanoparticle conjugates.

    PubMed

    Ciaurriz, Paula; Bravo, Ernesto; Hamad-Schifferli, Kimberly

    2014-01-15

    We investigate the activity of glucose oxidase (GOx) together with horseradish peroxidase (HRP) on carbon nanoparticles (CNPs). Because GOx activity relies on HRP, we probe how the arrangement of the enzymes on the CNPs affects enzymatic behavior. Colorimetric assays to probe activity found that the coupling strategy affects activity of the bienzyme-nanoparticle complex. GOx is more prone than HRP to denaturation on the CNP surface, where its activity is compromised, while HRP activity is enhanced when interfaced to the CNP. Thus, arrangements where HRP is directly on the surface of the CNP and GOx is not are more favorable for overall activity. Coverage also influenced activity of the bienzyme complex, but performing the conjugation in the presence of glucose did not improve GOx activity. These results show that the architecture of the assembly is an important factor in optimization of nanoparticle-protein interfaces. PMID:24231087

  9. Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

    PubMed Central

    Miyazaki, Tsuyoshi; Neff, Lynn; Tanaka, Sakae; Horne, William C.; Baron, Roland

    2003-01-01

    The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane–associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria. PMID:12615910

  10. Ascorbyl palmitate-loaded chitosan nanoparticles: characteristic and polyphenol oxidase inhibitory activity.

    PubMed

    Kim, Mi Kyung; Lee, Ji-Soo; Kim, Kwang Yup; Lee, Hyeon Gyu

    2013-03-01

    The aim of this study was to produce ascorbyl palmitate (AP)-loaded nanoparticles in order to inhibit polyphenol oxidase (PPO) in bananas. AP-loaded chitosan nanoparticles were prepared using acetic acid and citric acid (denoted as CS/AA and CS/CA nanoparticles, respectively). As the initial AP concentration increases, the particle size significantly decreases, and the zeta potential, entrapment and loading efficiency significantly increases. The PPO inhibitory activity of AP was effectively improved when AP was nano-encapsulated by chitosan compared to no encapsulation. These results suggest that chitosan nano-encapsulation can be used to enhance the PPO inhibitory activity of AP. PMID:23247266

  11. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  12. Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

    PubMed

    Sunita, Kumari; Habib, Maria; Kumar, P; Singh, Vinay Kumar; Husain, Syed Akhtar; Singh, D K

    2016-02-01

    Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4h LC50 of azadirachtin and allicin, respectively. PMID:26536397

  13. (/sup 11/C)clorgyline and (/sup 11/C)-L-deprenyl and their use in measuring functional monoamine oxidase activity in the brain using positron emission tomography

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1986-04-17

    This invention involves a new strategy for imaging the activity of the enzyme monoamine oxidase in the living body by using /sup 11/C-labeled enzyme inhibitors which bind irreversibly to an enzyme as a result of catalysis. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  14. Effects of special blue fluorescent light on hepatic mixed-function oxidase activity in the rat

    SciTech Connect

    Davis, D.R.; Yeary, R.A.; Randall, G.

    1981-01-01

    Phototherapy has been widely used in the treatment of neonatal hyperbilirubinemia. Recent reports, however, have indicated that fluorescent light may be toxic and mutagenic to mammalian cells. these findings suggest possible long-term side effects with the use of phototherapy. This study was undertaken to determine the effects of phototherapy on hepatic microsomal enzyme activity. The exposure of Sprague-Dawley and Gunn rats to special blue fluorescent light at an average irradiance of 1,200 microW/cm2 resulted in no significant changes in liver microsomal enzyme activity for aniline hydroxylase, p-nitroanisole-O-demethylase, ethylmorphine-N-demethylase, cytochrome c reductase or the quantity of cytochrome P-450. A significant decrease in aniline hydroxylase and p-nitroanisole-O-demethylase activity was observed when liver microsomes were exposed in vitro to special blue fluorescent light. Photoactivated bilirubin did not effect the activity of the mixed-function oxidase enzymes measured under the conditions of this study.

  15. Temperature dependence of the activity of polyphenol peroxidases and polyphenol oxidases in modern and buried soils

    NASA Astrophysics Data System (ADS)

    Yakushev, A. V.; Kuznetsova, I. N.; Blagodatskaya, E. V.; Blagodatsky, S. A.

    2014-05-01

    Under conditions of the global climate warming, the changes in the reserves of soil humus depend on the temperature sensitivities of polyphenol peroxidases (PPPOs) and polyphenol oxidases (PPOs). They play an important role in lignin decomposition, mineralization, and humus formation. The temperature dependence of the potential enzyme activity in modern and buried soils has been studied during incubation at 10 or 20°C. The experimental results indicate that it depends on the availability of the substrate and the presence of oxygen. The activity of PPOs during incubation in the absence of oxygen for two months decreases by 2-2.5 times, which is balanced by an increase in the activity of PPPOs by 2-3 times. The increase in the incubation temperature to 20°C and the addition of glucose accelerates this transition due to the more abrupt decrease in the activity of PPOs. The preincubation of the soil with glucose doubles the activity of PPPOs but has no significant effect on the activity of PPOs. The different effects of temperature on two groups of the studied oxidases and the possibility of substituting enzymes by those of another type under changing aeration conditions should be taken into consideration in predicting the effect of the climate warming on the mineralization of the soil organic matter. The absence of statistically significant differences in the enzymatic activity between the buried and modern soil horizons indicates the retention by the buried soil of some of its properties (soil memory) and the rapid restoration of high enzymatic activity during the preincubation.

  16. Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

    SciTech Connect

    Boxtel, Antonius L. van; Kamstra, Jorke H.; Fluitsma, Donna M.; Legler, Juliette

    2010-04-15

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

  17. Effect of afforestation on urate oxidase activity in two kinds of soils

    NASA Astrophysics Data System (ADS)

    Meysner, Teresa; Wojciech Szajdak, Lech

    2010-05-01

    Researches were carried out in soils under a 125-m-long the afforestation located in the Ko?cian Plain in Turew, which is a part of West Poland Lowland. Soil samples were taken from four chosen sites marked as Nos. 1, 2, 3 and 4 near wells. One part of this afforestation was allocated on mineral, whereas the second part was on mineral-organic soil. Times of sampling were from March to November in 2009 from the layer at 0-20 cm depth after removing leaf litter. Urate oxidase activity in soils was determined colorimetrically by measuring the absorbance at ?=293 nm. Urate oxidase is a homotetrameric enzyme containing four identical active sites situated at the interfaces between its four subunits. This enzyme catalyzes the oxidation of uric acid, a final product of purine catabolism to 5-hydroxyisourate, which is non-enzymatically transformed into allantoin, carbon dioxide and hydrogen peroxide. Uricase is also an essential enzyme in the ureide pathway, where nitrogen fixation occurs in the root nodules of legumes. Nitrogen heterocyclic compounds such as allantoin may serve as nitrogen sources or nitrogen transport compounds in plants that are not able to fix nitrogen. It has been estimated that heterocyclic nitrogen compounds represent about 30% of the reduced nitrogen in soils. These studies indicated that the flow of ground water was accompanied by an increase of uricase activity from 16 to 71% (from point 1 to point 2) in all periods of sampling in mineral soils. Similar trend was shown in mineral-organic soils. There was an increase of uricase activity from the point 3 to 4 and ranged from 13 to 37% similar to the direction of the flow of ground water. However, no significant differences of urate oxidase activity between two kinds of soils were observed. This study showed that the uricase activity ranged from 1.99 to 7.16 ?mol×h-1×g-1 in the mineral soils and from 1.79 to 8.36 ?mol×h-1×g-1. The study indicated an impact of the afforestation located on mineral and mineral-organic soils on the changes of uricase activity similar to the flow of ground water. This work was supported by a grant No. N N305 3204 36 founded by Polish Ministry of Education.

  18. A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

    PubMed

    Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

    2001-02-01

    Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus. PMID:11302173

  19. Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67phox

    PubMed Central

    Roos, Dirk; van Buul, Jaap D; Tool, Anton TJ; Matute, Juan D; Marchal, Christophe M; Hayee, Bu’Hussain; Köker, M Yavuz; de Boer, Martin; van Leeuwen, Karin; Segal, Anthony W; Pick, Edgar; Dinauer, Mary C

    2015-01-01

    Study background Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in CYBB, the X-linked gene that encodes gp91phox, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in NCF2, the gene that encodes p67phox, the activator subunit of the NADPH oxidase. Methods Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients’ NCF2 gene was expressed as Ala202Val-p67phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67phox from the cytosol of the patients’ neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification. Results The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67phox was clearly hypomorphic: substantial expression of p67phox protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20–70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67phox. Conclusion The mutation found in NCF2 is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67phox, resulting in reduced activation of gp91phox. PMID:25937994

  20. A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: Identification and mutagenesis of two conserved residues that are essential for enzyme activity

    SciTech Connect

    Wilks, H.M.; Timko, M.P.

    1995-01-31

    Protochlorophyllide reductase (NADPH:protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway. We have developed an expression system in which the protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme. By protein sequence comparisons, we have identified the plant protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases. Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity. A model of the enzyme reaction mechanism for light-dependent protochlorophyllide reduction is proposed. 33 refs., 5 figs.

  1. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays.

    PubMed

    Hayat, Akhtar; Cunningham, Jessica; Bulbul, Gonca; Andreescu, Silvana

    2015-07-23

    Nanomaterial-based enzyme mimics have attracted considerable interest in chemical analysis as alternative catalysts to natural enzymes. However, the conditions in which such particles can replace biological catalysts and their selectivity and reactivity profiles are not well defined. This work explored the oxidase like properties of nanoceria particles in the development of colorimetric assays for the detection of dopamine and catechol. Selectivity of the system with respect to several phenolic compounds, the effect of interferences and real sample analysis are discussed. The conditions of use such as buffer composition, selectivity, pH, reaction time and particle type are defined. Detection limits of 1.5 and 0.2?M were obtained with nanoceria for dopamine and catechol. The same assay could be used as a general sensing platform for the detection of other phenolics. However, the sensitivity of the method varies significantly with the particle type, buffer composition, pH and with the structure of the phenolic compound. The results demonstrate that nanoceria particles can be used for the development of cost effective and sensitive methods for the detection of these compounds. However, the selection of the particle system and experimental conditions is critical for achieving high sensitivity. Recommendations are provided on the selection of the particle system and reaction conditions to maximize the oxidase like activity of nanoceria. PMID:26231899

  2. Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase.

    PubMed

    Di Paolo, Maria Luisa; Lunelli, Michele; Fuxreiter, Monika; Rigo, Adelio; Simon, Istvan; Scarpa, Marina

    2011-04-01

    The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of K(m) and catalytic constant (k(c)) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed. PMID:21294844

  3. In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and -B Enzymatic Activity.

    PubMed

    Brooks, Allen F; Shao, Xia; Quesada, Carole A; Sherman, Phillip; Scott, Peter J H; Kilbourn, Michael R

    2015-12-16

    The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine, and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed to be an in vivo marker of neuroinflammation associated with Alzheimer's disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors or high-affinity reversibly binding inhibitors. As an alternative approach, we have investigated 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yield 1-[(11)C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl, and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brains. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain, the phenyl ether demonstrated MAO-A selectivity and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity. PMID:26393369

  4. Latent polyphenol oxidases from sago log (Metroxylon sagu): partial purification, activation, and some properties.

    PubMed

    Onsa, G H; bin Saari, N; Selamat, J; Bakar, J

    2000-10-01

    Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid. PMID:11052775

  5. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  6. Carnosine: effect on aging-induced increase in brain regional monoamine oxidase-A activity.

    PubMed

    Banerjee, Soumyabrata; Poddar, Mrinal K

    2015-03-01

    Aging is a natural biological process associated with several neurological disorders along with the biochemical changes in brain. Aim of the present investigation is to study the effect of carnosine (0.5-2.5?g/kg/day, i.t. for 21 consecutive days) on aging-induced changes in brain regional (cerebral cortex, hippocampus, hypothalamus and pons-medulla) mitochondrial monoamine oxidase-A (MAO-A) activity with its kinetic parameters. The results of the present study are: (1) The brain regional mitochondrial MAO-A activity and their kinetic parameters (except in Km of pons-medulla) were significantly increased with the increase of age (4-24 months), (2) Aging-induced increase of brain regional MAO-A activity including its Vmax were attenuated with higher dosages of carnosine (1.0-2.5?g/kg/day) and restored toward the activity that observed in young, though its lower dosage (0.5?g/kg/day) were ineffective in these brain regional MAO-A activity, (3) Carnosine at higher dosage in young rats, unlike aged rats significantly inhibited all the brain regional MAO-A activity by reducing their only Vmax excepting cerebral cortex, where Km was also significantly enhanced. These results suggest that carnosine attenuated the aging-induced increase of brain regional MAO-A activity by attenuating its kinetic parameters and restored toward the results of MAO-A activity that observed in corresponding brain regions of young rats. PMID:25450310

  7. Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits.

    PubMed

    González, E M; de Ancos, B; Cano, M P

    2000-11-01

    A partial characterization of peroxidase (POD) and polyphenol oxidase (PPO) activities in blackberry fruits is described. Two cultivars of blackberry (Wild and Thornless) were analyzed for POD and PPO activities. Stable and highly active POD and PPO extracts were obtained using insoluble poly(vinylpyrrolidone) and Triton X-100 in 0.05 M sodium phosphate, pH 7.5, buffer. Blackberry POD and PPO activities have a pH optimum of 6.5, in a reaction mixture of 0.2 M sodium phosphate. Optimal POD activity was found with 3% o-dianisidine. Maximum PPO activity was found with catechol (catecholase activity) followed by 4-methylcatechol. Polyacrylamide gel electrophoresis of blackberry extracts under non-denaturing conditions resolved in various bands. In the POD extracts of Wild fruits, there was only one band with a mobility of 0.12. In the Thornless POD extracts there were three well-resolved bands, with R(f) values of 0.63, 0.36, and 0.09. Both the Wild and Thornless blackberry cultivars produced a single band of PPO, with R(f) values of 0.1 for Wild and 0.06 for Thornless. PMID:11087502

  8. Activation of Polyphenol Oxidase in Dormant Wild Oat Caryopses by a Seed-Decay Isolate of Fusarium avenaceum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incubation of dormant wild oat (Avena fatua L., isoline M73) caryopses for 1 to 3 days with Fusarium avenaceum seed-decay isolate F.a.1 induced activity of the plant defense enzyme polyphenol oxidase (PPO). Both extracts and leachates obtained from F.a.1-treated caryopses had decreased abundance of ...

  9. Brief maternal deprivation of rats reduces hepatic mixed function oxidase activities

    SciTech Connect

    Vesell, E.S. ); Heubel, F.; Netter, K.J. )

    1989-01-01

    Deprivation of pups from mother and sibs for 3 min daily from day 5 today 41 of life reduced activities of 4 hepatic mixed function oxidases (MFO) expressed per mg protein in male rats compared to unhandled control rats. These decreases, though generally small, 22.4% and under, reached statistical significance for the substrates aminopyrine, benzphetamine and ethoxycoumarin. This handling procedure did not consistently affect the inductive response to phenobarbital. Previously ignored as a source of variability in response to xenobiotics, handling appears from these results to merit further investigation as such a factor in uninduced rats. Differences among rats in handling could contribute to large day-to-day variations in their metabolism of xenobiotics.

  10. Mia40 combines thiol oxidase and disulfide isomerase activity to efficiently catalyze oxidative folding in mitochondria.

    PubMed

    Koch, Johanna R; Schmid, Franz X

    2014-12-12

    Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein. PMID:25451030

  11. Purification of a unique glycoprotein that enhances phenol oxidase activity in scorpion (Heterometrus bengalensis) haemolymph.

    PubMed Central

    Datta, T K; Basu, P S; Datta, P K; Banerjee, A

    1989-01-01

    A monomeric glycoprotein (SGP) of Mr 32,000 was isolated to purity from scorpion (Heterometrus bengalensis) haemolymph by (NH4)2SO4 fractionation, chromatofocusing and h.p.l.c. The homogeneity of SGP is confirmed by polyacrylamide-gel electrophoresis. SGP is soluble in 100%-satd. (NH4)2SO4 solution. Needle-shaped crystals of SGP were obtained in an aqueous environment. The glycan part of the molecule contains arabinose, which does not commonly occur in animal glycoproteins. Amino acid analysis demonstrated a preponderance of glycine, tyrosine and glutamic acid. SGP enhances phenol oxidase (EC 1.14.18.1) activity. Images Fig. 3. Fig. 5. PMID:2504146

  12. Measurement of polyphenol oxidase activity using optical waveguide lightmode spectroscopy-based immunosensor.

    PubMed

    Kim, Namsoo; Kim, Woo-Yeon

    2015-02-15

    Polyphenol oxidase (PPO) is an important quality index during food processing involving heat-treatment and sensitive determination of PPO activity has been a critical concern in the food industry. In this study, a new measurement of PPO activity exploiting an optical waveguide lightmode spectroscopy-based immunosensor is presented using a polyclonal anti-PPO antibody that was immobilized in situ to the surface of a 3-aminopropyltriethoxysilane-treated optical grating coupler activated with glutaraldehyde. When analysed with a purified PPO fraction from potato tubers, a linear relationship was found between PPO activities of 0.0005607-560.7U/mL and the sensor responses obtained. The sensor was applicable to measurement of PPO activity in real samples that were prepared from potato tubers, grapes and Kimchi cabbage, and the analytical results were compared with those obtained by a conventional colorimetric assay measuring PPO activity. When tested for long-term stability, the sensor was reusable up to 10th day after preparation. PMID:25236218

  13. Blueberry polyphenol oxidase: Characterization and the kinetics of thermal and high pressure activation and inactivation.

    PubMed

    Terefe, Netsanet Shiferaw; Delon, Antoine; Buckow, Roman; Versteeg, Cornelis

    2015-12-01

    Partially purified blueberry polyphenol oxidase (PPO) in Mcllvaine buffer (pH=3.6, typical pH of blueberry juice) was subjected to processing at isothermal-isobaric conditions at temperatures from 30 to 80 °C and pressure from 0.1 to 700 MPa. High pressure processing at 30-50 °C at all pressures studied caused irreversible PPO activity increase with a maximum of 6.1 fold increase at 500 MPa and 30 °C. Treatments at mild pressure-mild temperature conditions (0.1-400 MPa, 60 °C) also caused up to 3 fold PPO activity increase. Initial activity increase followed by a decrease occurred at relatively high pressure-mild temperature (400-600 MPa, 60 °C) and mild pressure-high temperature (0.1-400 MPa, 70-80 °C) combinations. At temperatures higher than 76 °C, monotonic decrease in PPO activity occurred at 0.1 MPa and pressures higher than 500 MPa. The activation/inactivation kinetics of the enzyme was successfully modelled assuming consecutive reactions in series with activation followed by inactivation. PMID:26041182

  14. Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

    PubMed

    Nagler, L G; Vartanyan, L S

    1976-03-18

    Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD. PMID:1260010

  15. Polyphenol oxidase activity in subcellular fractions of tall fescue contaminated by polycyclic aromatic hydrocarbons.

    PubMed

    Ling, Wanting; Lu, Xiaodan; Gao, Yanzheng; Liu, Juan; Sun, Yandi

    2012-01-01

    Understanding enzyme responses to contamination with persistent organic pollutants (POPs) is a key step in the elucidation of POP metabolic mechanisms in plants. However, there is little information available on enzyme activity in subcellular fractions of POP-contaminated plants. To our knowledge, this is the first study to investigate the activities of polyphenol oxidase (PPO) in cell fractions of plants under contamination stress from polycyclic aromatic hydrocarbons (PAHs) using a greenhouse batch technique. Three parameters, E(cell), E(cell-n), and P(cell), denoting the amount of PPO activity, cell fraction content-normalized PPO activity, and proportion of PPO activity in each cell fraction, respectively, were used in this study. Contamination with phenanthrene, as a representative PAH, at a relatively high level (>0.23 mg L?¹) in culture solution generally stimulated PPO activity in tall fescue (Festuca arundinacea Schreb.) roots and shoots and their cellular fractions. The amount and distribution proportion of PPO activity in each cell fraction of phenanthrene-contaminated roots and shoots were (in descending order): cell solution > > cell wall > cell organelles. Cell solution was the dominant storage domain of PPO activity and contributed 84.0 and 82.8% of PPO activity in roots and shoots, respectively. The cell wall had the highest density of PPO activity in roots and shoots, based on the highest cell fraction content normalized PPO activity in this cell fraction. Our results provide new information on enzyme responses in plant intracellular fractions to xenobiotic POPs and fundamental information on within-plant POP metabolic mechanisms. PMID:22565262

  16. Peroxygenase and Oxidase Activities of Dehaloperoxidase-Hemoglobin from Amphitrite ornata

    PubMed Central

    2015-01-01

    The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5?-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5?-Br2-indigo, and together suggest a newly identified oxidase activity for DHP. PMID:24791647

  17. Potato and mushroom polyphenol oxidase activities are differently modulated by natural plant extracts.

    PubMed

    Kuijpers, Tomas F M; van Herk, Teunie; Vincken, Jean-Paul; Janssen, Renske H; Narh, Deborah L; van Berkel, Willem J H; Gruppen, Harry

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones. PMID:24344979

  18. Neurobehavioral evaluation of Reln-rl-orl mutant mice and correlations with cytochrome oxidase activity.

    PubMed

    Lalonde, R; Hayzoun, K; Derer, M; Mariani, J; Strazielle, C

    2004-07-01

    The Reln-rl-orl mutation is characterized by a marked deficit in cerebellar granule cell and Purkinje cell number as well as ectopias in cerebellum, hippocampus, and neocortex. By comparison to Balb/c controls, Reln-rl-orl mutants did not alternate spontaneously in a T-maze and were deficient for visuomotor guidance in a water maze. Despite cerebellar ataxia and motor coordination impairments on stationary beam, coat-hanger, and rotorod tests, the horizontal motor activity of Reln-rl-orl mutants was not reduced in an open-field. The elevated cytochrome oxidase (CO) activity in Purkinje cells and the reduced CO activity in the roof nuclei (interpositus and dentate) of the mutants were associated with poor performance on the small stationary beam. In addition, deficient CO activity of the granular layer of the motor cortex was associated with shorter latencies before falling from the larger stationary beam and a lower number of rears in the open-field. Conversely, elevated CO activity in the polymorphic layer of primary somatosensory cortex was congruent with higher latencies before falling from the same apparatus, indicating functional compensation. PMID:15196778

  19. Peroxygenase and oxidase activities of dehaloperoxidase-hemoglobin from Amphitrite ornata.

    PubMed

    Barrios, David A; D'Antonio, Jennifer; McCombs, Nikolette L; Zhao, Jing; Franzen, Stefan; Schmidt, Andreas C; Sombers, Leslie A; Ghiladi, Reza A

    2014-06-01

    The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5'-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5'-Br2-indigo, and together suggest a newly identified oxidase activity for DHP. PMID:24791647

  20. Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

    PubMed Central

    Arora, Y K; Sinha, R C

    1985-01-01

    Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants. Images PMID:2997132

  1. Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation.

    PubMed

    Miletti, Teresa; Farber, Patrick J; Mittermaier, Anthony

    2011-09-01

    We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1? ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors. PMID:21947916

  2. Monoamine oxidase activity and tri-iodothyronine level in violent offenders with early behavioural problems.

    PubMed

    Eklund, Jenny; Alm, Per Olof; af Klinteberg, Britt

    2005-01-01

    The focus is on evaluating the relationships between early behavioural problems and biochemical variables at adult age and their significance for early criminality and violent behaviour in a life perspective. In the present study, using prospective longitudinal data, a sample of males with a history of early criminal behaviour and male controls (n = 103) were investigated concerning (1) teacher-rated behaviours at age 11-14 years; (2) platelet monoamine oxidase (MAO) activity and tri-iodothyronine (T(3)) level at adult age; (3) registered early criminality (11-14 years); (4) records of violent offending up to age 35 years, and (5) interview data on smoking. The main finding was that a combined risk level pattern of low MAO activity and high T(3) level was found significantly more frequently than expected in violent offenders with an early behavioural risk pattern. Furthermore, there was a significant interaction effect between early attention difficulties and smoking on MAO activity, as well as an effect by smoking on MAO activity. The findings are discussed in terms of the possible influence of biological vulnerability to certain behaviours, which in combination with possible childhood stress, enhance the risk for antisocial behaviours and subsequent violence. PMID:16113590

  3. Decreased ubiquinone availability and impaired mitochondrial cytochrome oxidase activity associated with statin treatment.

    PubMed

    Duncan, Andrew J; Hargreaves, Iain P; Damian, Maxwell S; Land, John M; Heales, Simon J R

    2009-01-01

    In order to investigate the potential involvement of mitochondrial electron transport chain (ETC) dysfunction in myotoxicity associated with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) treatment, assessment was made of ETC activity and ubiquinone status in two patients experiencing myopathy following treatment with simvastatin (40 mg/day) and cyclosporin (patient 1) and simvastatin (40 mg/day) and itraconazole (patient 2). Analysis of skeletal muscle biopsies revealed a decreased ubiquinone status (77 and 132; reference range: 140-580 pmol/mg) and cytochrome oxidase (complex IV) activity (0.006 and 0.007 reference range: 0.014-0.034). To assess statin treatment in the absence of possible pharmacological interference from cyclosporin or itraconazole, primary astrocytes were cultured with lovastatin (100 microM). Lovastatin treatment resulted in a decrease in ubiquinone (97.9 +/- 14.9; control: 202.9 +/- 18.4 pmol/mg; p < 0.05), and complex IV activity (0.008 +/- 0.001; control: 0.011 +/- 0.001; p < 0.05) relative to control. These data, coupled with the patient findings, indicate a possible association between statin treatment, decreased ubiquinone status, and loss of complex IV activity. PMID:19778232

  4. Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle.

    PubMed

    Fajardo, Val Andrew; McMeekin, Lauren; Saint, Caitlin; LeBlanc, Paul J

    2015-04-01

    Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2?6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2?6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2?6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p = 0.009, R(2) = 0.21). Specifically, our results suggest that CL 18:2?6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle. PMID:25727371

  5. Methadone, monoamine oxidase, and depression: opioid distribution and acute effects on enzyme activity

    SciTech Connect

    Kaufmann, C.A.; Kreek, M.J.; Raghunath, J.; Arns, P.

    1983-09-01

    Narcotic withdrawal is often accompanied by an atypical depression which responds to resumption of narcotics. It was hypothesized that methadone might exert its antidepressant effects through monoamine oxidase (MAO) inhibition. The current study examined /sub 3/H-methadone distribution in rat brain and effects on regional MAO activity with acute doses (2.5 mg/kg) which approximate those found during chronic methadone maintenance in man. Limbic areas (amygdala, basomedial hypothalamus, caudate-putamen, hippocampus, preoptic nucleus), as well as pituitary and liver were assayed for MAO activity and methadone concentration. MAO activities did not differ significantly in acute methadone or saline-treated cage-mates at 1 or 24 hr. The concentrations of methadone at 1 hr ranged between 17 and 223 ng/100 mg wet wt tissue in the preoptic nucleus and pituitary, respectively. No significant correlation was found between change in MAO activity (MAO methadone/MAO saline) and methadone concentration in any region at 1 or 24 hr. This study does not support the hypothesis that methadone acts as an antidepressant through MAO inhibition, at least not following acute administration of this exogenous opioid.

  6. Rac1-Mediated Activation of Mineralocorticoid Receptor in Pressure Overload-Induced Cardiac Injury.

    PubMed

    Ayuzawa, Nobuhiro; Nagase, Miki; Ueda, Kohei; Nishimoto, Mitsuhiro; Kawarazaki, Wakako; Marumo, Takeshi; Aiba, Atsu; Sakurai, Takayuki; Shindo, Takayuki; Fujita, Toshiro

    2016-01-01

    There is increasing evidence for a crucial role of aberrant mineralocorticoid receptor (MR) activation in heart failure, with clinical studies showing beneficial effects of MR blockade. However, the mechanisms of MR activation in heart failure remain unclear. In this study, we observed that the small GTPase Rac1 contributes to myocardial MR activation, whereas Rac1-MR pathway activation leads to cardiac dysfunction. Mouse hearts subjected to chronic pressure overload induced by transverse aortic constriction showed Rac1 activation and increased nuclear accumulation of MR and expression of MR target genes, suggesting MR activation. Pharmacological inhibition of Rac1 and heterozygous deletion of Rac1 in cardiomyocytes suppressed Rac1-induced MR signaling and reduced NADPH oxidase 4 gene induction and reactive oxygen species overproduction, which attenuated transverse aortic constriction-induced cardiac hypertrophy and dysfunction. Consistently, treatment with the selective MR antagonist eplerenone blocked transverse aortic constriction-induced MR signaling and NADPH oxidase 4 gene upregulation, which improved cardiac hypertrophy and dysfunction. These findings suggest that Rac1-MR pathway activation in the myocardium is involved in development of heart failure induced by pressure load via recruitment of the responsible isoform of NADPH oxidase. Thus, the cardiac Rac1-MR-NADPH oxidase 4 pathway may be a therapeutic target for treatment of the pressure-overloaded heart. PMID:26527051

  7. Transgenic tobacco (Nicotiana tabacum L.) plants with increased expression levels of mitochondrial NADP+-dependent isocitrate dehydrogenase: evidence implicating this enzyme in the redox activation of the alternative oxidase.

    PubMed

    Gray, Gordon R; Villarimo, Alicia R; Whitehead, Carmen L; McIntosh, Lee

    2004-10-01

    Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction. One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 1.1.1.42), although the precise role of this enzyme is not yet known. Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX). We have partially purified mtICDH from tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS-PAGE, which was verified by peptide sequencing. In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak. Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity. In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type. When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line. These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX. PMID:15564525

  8. Purification, characterization and antibacterial activity of L-amino acid oxidase from Cerastes cerastes.

    PubMed

    Hanane-Fadila, Ziad-Meziane; Fatima, Laraba-Djebari

    2014-08-01

    Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram-positive and Gram-negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An L-amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS-PAGE. LC-MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin-resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 ?g/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on L-leucine at pH 8.0 and 20°C were Km = 0.06 mmol and Vmax = 164 mmol/min. PMID:24817275

  9. Xanthine oxidase inhibitory activity and hypouricemic effect of aspalathin from unfermented rooibos.

    PubMed

    Kondo, Makoto; Hirano, Yoshiaki; Nishio, Masahiro; Furuya, Yutaka; Nakamura, Hiromichi; Watanabe, Tsuyoshi

    2013-12-01

    Rooibos is rich in flavonoids such as aspalathin, which is a unique C-glycosyl dihydrochalcone, that is used as a traditional herbal tea. This study was designed to evaluate the in vitro xanthine oxidase (XOD) inhibitory activity of the aspalathin-rich fraction (ARF) and purified aspalathin from rooibos. The hypouricemic effects of the ARF and aspalathin on hyperuricemic mice were also assessed. The ARF was prepared from aqueous extract of unfermented rooibos leaves and stems, and it was collected by column chromatography; the aspalathin content in this fraction was 21.4%. The ARF and aspalathin inhibited XOD in a dose-dependent manner. The concentrations of the ARF and aspalathin required to inhibit XOD at 50% (IC50 ) were 20.4 ?g/mL (4.4 ?g/mL aspalathin equivalents) and 4.5 ?g/mL, respectively. Lineweaver-Burk plot analysis indicated that aspalathin was a competitive inhibitor of XOD, and the inhibition constant (Ki) was 3.1 ?M. In hyperuricemic mice induced by inosine-5'-monophosphate, treatment with the ARF and aspalathin significantly suppressed the increased plasma uric acid level in a dose-dependent manner. The suppressed plasma uric acid level in mice could be attributed to the XOD inhibitory activity of the ARF and aspalathin. Further study is required to determine the effect of aspalathin or its metabolites on XOD activity in vivo. PMID:24261664

  10. Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

    PubMed

    Cheng, Xi; Huang, Xingjian; Liu, Siyu; Tang, Mi; Hu, Wanfeng; Pan, Siyi

    2014-07-01

    Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity. PMID:24845377

  11. Cell surface NADH oxidase activity of brine shrimp oscillates with a period of 25 min and is entrained by light.

    PubMed

    Chalko, C J; Morré, D M; Morré, D J

    2000-01-01

    Plants have a surface NADH oxidase that measures time by oscillating with a 24-min period. The period is synchronized by light. With plants, a new maximum is observed exactly 12 min after the beginning of the light exposure. These experiments were to determine if animals exhibited a cell surface NADH oxidase having a similar periodicity and to answer the question, does the periodicity in animals respond to light? Using brine shrimp as a model, the findings show that plants and animals exhibit similar oscillating NADH oxidase activity and that the periodicity in this invertebrate animal does respond to light. Brine shrimp were grown for two to three days and transferred to darkness for 45 min. After return to light for one min, NADH was added and measurements of NADH oxidation were recorded over 50 min. The brine shrimp exhibited a cell surface NADH oxidase that oscillated with a period of 25 min. After being subjected to light, the brine shrimp showed a new maximum in NADH oxidation between 12 to 13 min after the beginning of the light exposure and again at 37 min and at 25 min intervals thereafter. The findings demonstrate that the periodic oscillations in NADH oxidation of brine shrimp are light entrainable. PMID:10894092

  12. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  13. A high-performance-liquid-chromatographic method for the assay of coproporphyrinogen oxidase activity in rat liver.

    PubMed Central

    Li, F; Lim, C K; Peters, T J

    1986-01-01

    An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy. PMID:3814086

  14. Xanthine oxidase inhibitory activity of the methanolic extracts of selected Jordanian medicinal plants

    PubMed Central

    Hudaib, Mohammad M.; Tawaha, Khaled A.; Mohammad, Mohammad K.; Assaf, Areej M.; Issa, Ala Y.; Alali, Feras Q.; Aburjai, Talal A.; Bustanji, Yasser K.

    2011-01-01

    Background: The search for novel xanthine oxidase (XO) inhibitors with a higher therapeutic activity and fewer side effects are desired not only to treat gout but also to combat various other diseases associated with the XO activity. At present, the potential of developing successful natural products for the management of XO-related diseases is still largely unexplored. In the present study, we have screened the methanolic extracts of various Jordanian medicinal plants for their XO inhibitory activities using an optimized protocol. Materials and Methods: The methanolic extracts of 23 medicinal plants, belonging to 12 families, were tested in vitro, at 200 ?g/ml concentrations, for their XO inhibitory potential. The dose-dependent inhibition profiles of the most active plants were further evaluated by estimating the IC50 values of their corresponding extracts. Results: Six plants were found most active (% inhibition more than 39%). These plants are Salvia spinosa L. (IC50 = 53.7 ?g/ml), Anthemis palestina Boiss. (168.0 ?g/ml), Chrysanthemum coronarium L. (199.5 ?g/ml), Achillea biebersteinii Afansiev (360.0 ?g/ml), Rosmarinus officinalis L. (650.0 ?g/ml), and Ginkgo biloba L. (595.8 ?g/ml). Moreover, four more plants, namely Lavandula angustifolia Mill. (28.7% inhibition), Helianthemum ledifolium (L.) Mill. (28.4%), Majorana syriaca (L.) Kostel. (25.1%), and Mentha spicata L. (22.5%) showed a XO inhibitory activity in the range of 22–30%. Conclusion: The study showed that many of the tested plant species are potential sources of natural XO inhibitors that can be developed, upon further investigation, into successful herbal drugs for treatment of gout and other XO-related disorders. PMID:22262935

  15. Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress.

    PubMed

    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-02-01

    Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 ?M AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed. PMID:24246127

  16. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  17. Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

    PubMed

    Boschi-Muller, Sandrine; Branlant, Guy

    2014-12-01

    Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form. PMID:25108804

  18. Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6

    PubMed Central

    Schüll, S; Günther, S D; Brodesser, S; Seeger, J M; Tosetti, B; Wiegmann, K; Pongratz, C; Diaz, F; Witt, A; Andree, M; Brinkmann, K; Krönke, M; Wiesner, R J; Kashkar, H

    2015-01-01

    Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16?:?0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction. PMID:25766330

  19. Parameters That Enhance the Bacterial Expression of Active Plant Polyphenol Oxidases

    PubMed Central

    Dirks-Hofmeister, Mareike E.; Kolkenbrock, Stephan; Moerschbacher, Bruno M.

    2013-01-01

    Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26°C). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a vmax of 0.48±0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes. PMID:24204791

  20. Parameters that enhance the bacterial expression of active plant polyphenol oxidases.

    PubMed

    Dirks-Hofmeister, Mareike E; Kolkenbrock, Stephan; Moerschbacher, Bruno M

    2013-01-01

    Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26 °C). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v(max) of 0.48 ± 0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes. PMID:24204791

  1. Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes.

    PubMed

    Massey, V; Schopfer, L M; Nishino, T; Nishino, T

    1989-06-25

    The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase. PMID:2732238

  2. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity

    PubMed Central

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor.

  3. Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

    SciTech Connect

    Silanikove, Nissim Shapiro, Fira; Leitner, Gabriel

    2007-11-23

    The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T{sub 1/2} {approx} 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.

  4. CO-dynamics in the active site of cytochrome c oxidase.

    PubMed

    Soloviov, Maksym; Meuwly, Markus

    2014-04-14

    The transfer of CO from heme a3 to the Cu(B) site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the Cu(B) site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ?90% of the ligands are found to rebind to either the Cu(B) (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet. PMID:24735320

  5. Magnetic colorimetric immunoassay for human interleukin-6 based on the oxidase activity of ceria spheres.

    PubMed

    Peng, Juan; Guan, Jufang; Yao, Huiqin; Jin, Xiaoyong

    2016-01-01

    A novel magnetic colorimetric immunoassay strategy was designed for sensitive detection of human interleukin-6 (IL-6) using ceria spheres as labels. Ceria spheres showed excellent oxidase activity, which can directly catalyze the oxidation of substrate o-phenylenediamine (OPD) to a stable yellow product, 2,3-diaminophenazine (oxOPD). The absorbance of oxOPD was recorded to reflect the level of IL-6. The relatively mild conditions made the immunoassay strategy more robust, reliable, and easy. A linear relationship between absorbance intensity and the logarithm of IL-6 concentrations was obtained in the range of 0.0001-10 ng mL(-1) with a detection limit of 0.04 pg mL(-1) (S/N = 3). The colorimetric immunoassay exhibited high sensitivity and specificity for the detection of IL-6. This immunoassay has been successfully applied in the detection of IL-6 in serum samples and can be readily extended toward the on-site monitoring of cancer biomarkers in serum samples. PMID:26416691

  6. Successful Treatment of Intracranial Glioblastoma Xenografts With a Monoamine Oxidase B-Activated Pro-Drug

    PubMed Central

    Sharpe, Martyn A.; Livingston, Andrew D.; Gist, Taylor L.; Ghosh, Pardip; Han, Junyan; Baskin, David S.

    2015-01-01

    The last major advance in the treatment of glioblastoma multiforme (GBM) was the introduction of temozolomide in 1999. Treatment with temozolomide following surgical debulking extends survival rate compared to radiotherapy and debulking alone. However, virtually all glioblastoma patients experience disease progression within 7 to 10 months. Although many salvage treatments, including bevacizumab, rechallenge with temozolomide, and other alkylating agents, have been evaluated, none of these clearly improves survival. Monoamine oxidase B (MAOB) is highly expressed in glioblastoma cell mitochondria, and mitochondrial function is intimately tied to treatment-resistant glioblastoma progression. These glioblastoma properties provide a strong rationale for pursuing a MAOB-selective pro-drug treatment approach that, upon drug activation, targets glioblastoma mitochondria, especially mitochondrial DNA. MP-MUS is the lead compound in a family of pro-drugs designed to treat GBM that is converted into the mature, mitochondria-targeting drug, P+-MUS, by MAOB. We show that MP-MUS can successfully kill primary gliomas in vitro and in vivo mouse xenograft models. PMID:26501110

  7. Platelet monoamine oxidase (MAO) activity: evidence for a single major locus.

    PubMed Central

    Rice, J; McGuffin, P; Goldin, L R; Shaskan, E G; Gershon, E S

    1984-01-01

    Monoamine oxidase (MAO), a mitochondrial enzyme involved in the degradation of biogenic amines, has been associated with psychiatric morbidity. Although twin and family studies have indicated that MAO activity is familial, the exact mode of transmission is unclear. We performed segregation analysis on 154 nuclear families containing 419 individuals using the mixed model, which allows for a single major locus with a polygenic background. We were able to reject a dominant and additive locus with or without a heritable background and a recessive locus without background. The acceptable models were: (1) a codominant model without background where the mean of the heterozygote distribution was 30% of the distance from the low to the high homozygote distributions, and (2) a recessive locus with heritable background. In both cases, the gene frequency for the high-MAO allele is approximately .25--at odds with suggestions that low-MAO represents a genetic marker for a disorder such as schizophrenia with a lifetime risk of only 0.85%. To ensure that results were not artifacts from a familial, skewed distribution, the data were also analyzed after power transformation. In addition, hypotheses were tested using both the joint and conditional likelihoods to examine for possible misspecification of the model with respect to intergenerational differences. Finally, we allowed for non-Mendelian transmission probabilities to provide another class of alternatives against which to test the hypothesis of a major locus. All these approaches provided additional confirmation for the presence of a major locus segregating within these families. PMID:6695924

  8. Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

    PubMed

    Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

    2014-12-01

    In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors. PMID:24246090

  9. Rapid conversion to high xanthine oxidase activity in viable Kupffer cells during hypoxia.

    PubMed Central

    Wiezorek, J S; Brown, D H; Kupperman, D E; Brass, C A

    1994-01-01

    It has been widely postulated that the central mechanism of hepatic reperfusion injury involves the conversion, during ischemia, of the enzyme xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD). However, this theory has been questioned because (a) XDH to XOD conversion in whole liver occurs very slowly; (b) the cellular distribution of XDH/XOD is unclear; and (c) the direct demonstration of XDH to XOD conversion in viable cells is lacking. In this paper, we address all three issues by measuring XDH to XOD conversion and cell viability in purified populations of hepatic endothelial cells (EC), Kupffer cells (KC), and hepatocytes (HEP). Although XDH/XOD activity on a cellular basis was greater in hepatocytes (0.92 +/- 0.12 mU/10(6) cells) than ECs (0.03 +/- 0.01) or KCs (0.12 +/- 0.04), XDH + XOD specific activity was similar in all three cell types (HEP 1.85 +/- 0.10 U/g protein; EC 1.69 +/- 0.54; KC 2.30 +/- 0.22). Over 150 min of warm (37 degrees C) or 24 h of cold (4 degrees C) hypoxia, percent XOD activity increased slowly in ECs, from 21 +/- 2% (basal) to 39 +/- 3% (warm) and 49 +/- 5% (cold) and in HEPs (29 +/- 2% to 38 +/- 3% and 49 +/- 2%), but converted significantly faster in KCs (28 +/- 3% to 91 +/- 7% and 94 +/- 4%). The dramatic changes in Kupffer cell XOD during cold hypoxia occurred despite only minor changes in cell viability. When hypoxic KCs were reoxygenated after 16 h of cold hypoxia, there was a marked increase in cell death that was significantly blocked by allopurinol. These data suggest that significant conversion to the free radical-producing state occurs within viable KCs, and that Kupffer cell XOD may play an important role in mediating reperfusion injury in the liver. PMID:7989578

  10. Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage.

    PubMed

    Páramo, Blanca; Montiel, Teresa; Hernández-Espinosa, Diego R; Rivera-Martínez, Marlene; Morán, Julio; Massieu, Lourdes

    2013-11-01

    The mechanisms leading to neuronal death during glucose deprivation have not been fully elucidated, but a role of oxidative stress has been suggested. In the present study we have investigated whether the production of reactive oxygen species during glucose deprivation, contributes to the activation of calpain, a calcium-dependent protease involved in neuronal injury associated with brain ischemia and cerebral trauma. We have observed a rapid activation of calpain, as monitored by the cleavage of the cytoskeletal protein ?-spectrin, after glucose withdrawal, which is reduced by inhibitors of xanthine oxidase, phospholipase A2 and NADPH oxidase. Results suggest that phospholipase A2 and NADPH oxidase contribute to the early activation of calpain after glucose deprivation. In particular NOX2, a member of the NADPH oxidase family is involved, since reduced stimulation of calpain activity is observed after glucose deprivation in hippocampal slices from transgenic mice lacking a functional NOX2. We observed an additive effect of the inhibitors of xanthine oxidase and phospholipase A2 on both ROS production and calpain activity, suggesting a synergistic action of these two enzymes. The present results provide new evidence showing that reactive oxygen species stimulate calpain activation during glucose deprivation and that this mechanism is involved in neuronal death. PMID:23994487

  11. Antiproliferative activity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

    PubMed

    Li Lee, Mui; Chung, Ivy; Yee Fung, Shin; Kanthimathi, M S; Hong Tan, Nget

    2014-04-01

    King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 ?g/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 ?g/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours. PMID:24118879

  12. Pulsed EPR Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

    PubMed Central

    Klein, Eric L.; Belaidi, Abdel Ali; Raitsimring, Arnold M.; Davis, Amanda C.; Krämer, Tobias; Astashkin, Andrei V.; Neese, Frank; Schwarz, Günter; Enemark, John H.

    2014-01-01

    Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ? 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of the density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO variant are dominated by the ‘inter-doublet’ transition arising from the strong nuclear quadrupole interaction, as also occurs for the 33S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al., Inorg. Chem. 2012, 51, 1408 – 1418]. The estimated experimental hfi and nqi parameters for 33S (aiso = 3 MHz and e2Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two 33S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V). PMID:24387640

  13. Microfluidic Devices Integrating Microcavity Surface-Plasmon-Resonance Sensors: Glucose Oxidase Binding-Activity Detection

    PubMed Central

    Amarie, Dragos; Alileche, Abdelkrim; Dragnea, Bogdan; Glazier, James A.

    2010-01-01

    We have developed miniature (?1 ?m diameter) microcavity surface-plasmon-resonance sensors (MSPRS), integrated them with microfluidics and tested their sensitivity to refractive-index changes. We tested their biosensing capability by distinguishing the interaction of glucose oxidase (Mr 160 kDa) with its natural substrate (?-D-glucose, Mr 180 Da) from its interactions with non-specific substrates (L-glucose, D-mannose and 2-deoxy-D-glucose). We ran the identical protocol we had used with the MSPRS on a Biacore 3000 instrument using their bare gold chip. Only the MSPRS was able to detect ?-D-glucose binding to glucose oxidase. Each MSPRS can detect the binding to its surface of fewer than 35,000 glucose-oxidase molecules (representing 9.6 fg or 60 zmol of protein), about 106 times fewer than classical surface-plasmon-resonance biosensors. PMID:19968248

  14. Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration

    E-print Network

    Mander, Palwinder K.; Brown, Guy C.

    2005-09-12

    stream_source_info 1742-2094-2-20.pdf.txt stream_content_type text/plain stream_size 60957 Content-Encoding UTF-8 stream_name 1742-2094-2-20.pdf.txt Content-Type text/plain; charset=UTF-8 ral ssBioMed Cent... inflammation may contribute to many CNS pathologies including Alzheimer's, Parkinson's and motor neuron diseases, mul- tiple sclerosis, meningitis, AIDS dementia, strokes, trauma and normal brain ageing [6,7]. It is therefore important to understand...

  15. Mechanisms of Oxidase and Superoxide Dismutation-like Activities of Gold, Silver, Platinum, and Palladium, and Their Alloys: A General Way to the Activation of Molecular Oxygen.

    PubMed

    Shen, Xiaomei; Liu, Wenqi; Gao, Xuejiao; Lu, Zhanghui; Wu, Xiaochun; Gao, Xingfa

    2015-12-23

    Metal and alloy nanomaterials have intriguing oxidase- and superoxide dismutation-like (SOD-like) activities. However, origins of these activities remain to be studied. Using density functional theory (DFT) calculations, we investigate mechanisms of oxidase- and SOD-like properties for metals Au, Ag, Pd and Pt and alloys Au4-xMx (x = 1, 2, 3; M = Ag, Pd, Pt). We find that the simple reaction-dissociation of O2-supported on metal surfaces can profoundly account for the oxidase-like activities of the metals. The activation (Eact) and reaction energies (Er) calculated by DFT can be used to effectively predict the activity. As verification, the calculated activity orders for series of metal and alloy nanomaterials are in excellent agreement with those obtained by experiments. Briefly, the activity is critically dependent on two factors, metal compositions and exposed facets. On the basis of these results, an energy-based model is proposed to account for the activation of molecular oxygen. As for SOD-like activities, the mechanisms mainly consist of protonation of O2(•-) and adsorption and rearrangement of HO2(•) on metal surfaces. Our results provide atomistic-level insights into the oxidase- and SOD-like activities of metals and pave a way to the rational design of mimetic enzymes based on metal nanomaterials. Especially, the O2 dissociative adsorption mechanism will serve as a general way to the activation of molecular oxygen by nanosurfaces and help understand the catalytic role of nanomaterials as pro-oxidants and antioxidants. PMID:26642084

  16. Evidence for a common regulation in the activation of a polyphenol oxidase by trypsin and sodium dodecyl sulfate.

    PubMed

    Gandía-Herrero, Fernando; Jiménez-Atiénzar, Mercedes; Cabanes, Juana; García-Carmona, Francisco; Escribano, Josefa

    2005-06-01

    Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3 , showed a value of 0.03 s(-1). The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE. PMID:16006247

  17. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  18. Purification of sulfide oxidase from rat liver 

    E-print Network

    Pu, Lixia

    1994-01-01

    The present study represents an initial investigative effort to purify sulfide oxidase from rat liver. Two methods to determine sulfide oxidase activity have been established and both are based on measuring substrate ...

  19. Patterns of cytochrome oxidase activity in the visual cortex of a South American opossum (Didelphis marsupialis aurita).

    PubMed

    Martinich, S; Rosa, M G; Rocha-Miranda, C E

    1990-01-01

    The normal pattern of cytochrome oxidase (CO) activity in the posterior cortical areas of the South American opossum (Didelphis marsupialis aurita) was assessed both in horizontal sections of flattened cortices and in transversal cortical sections. The tangential distribution of CO activity was uniformly high in the striate cortex. In the peristriate region alternating bands of dense and weak staining occupied all the cortical layers with the exception of layer I. This observation suggests the existence of a functional segregation of visual processing in the peristriate cortex of the opossum similar to that present in phylogenetically more recent groups. PMID:1966241

  20. Cytochrome b[sub 558]-negative, autosomal recessive chronic granulomatous disease: Two new mutations in the cyctochrome b[sub 558] light chain of the NADPH oxidase (p22-phox)

    SciTech Connect

    Boer, M. de; Klein, A. de; Weening, R.S.; Roos, D. ); Hossle, J.P.; Seger, R.; Corbeel, L.

    1992-11-01

    Chronic granulomatous disease (CGD) is characterized by the failure of activated phagocytes to generate superoxide. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b[sub 558] (gp91-phox). Patients with a rare autosomal recessive form of CGD have mutations in the gene for the alpha-subunit of this cytochrome (p22-phox). Usually, this leads to the absence of cytochrome b[sub 558] in the phagocytes (A22[sup 0] CGD). The authors studied the molecular defect in five European patients from three unrelated families with this type of CGD. P22-phox mRNA was reverse-transcribed, and the coding region was amplified by PCR in one fragment and sequenced. Three patients from one family, with parents that were first cousins, were homozygous for a single base substitution (G-297[yields]A) resulting in a nonconservative amino acid change (Arg-90-Gln). This mutation was previously found in a compound heterozygote A22[sup 0] CGD patient. Another patient, also from first-cousin parents, was homozygous for an A-309[yields]G mutation in the open reading frame that predicts a nonconservative amino acid replacement (His-94[yields]Arg). The fifth patient was also born from a first-cousin marriage and was shown to be homozygous for the absence of exon 4 from the cDNA. In this patient, a G[yields]A substitution was found at position 1 one intron 4 in the genomic DNA. Therefore, the absence of exon 4 in the cDNA of this patient is due to a splicing error. Two additional polymorphisms were also identified - one silent mutation in the open reading frame (G-508[l arrow][r arrow]A) and one A-640[l arrow][r arrow]G mutation in the 3'untranslated region of the p22-phox mRNA. This last mutation destroys a DraIII recognition site and is therefore potentially useful for RFLP analysis of CGD families. 22 refs., 4 figs., 2 tabs.

  1. Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  2. Dietary fat source influences neuronal mitochondrial monoamine oxidase activity and macronutrient selection in rats.

    PubMed

    Crane, S B; Greenwood, C E

    1987-05-01

    We previously reported that qualitative changes in dietary fat influence certain monoaminergic mediated behaviours such as pain sensitivity and thermoregulation in a cold environment after an amphetamine challenge. The purpose of this study was to further explore the behavioural consequences of alterations in dietary fat intake by examining another behaviour known to be mediated by the monoamines--food intake regulation--and to begin investigating a biochemical link between dietary fat composition and behaviour. Rats were stabilized to 20% (w/w) soybean oil (SBO) or lard diets for 10 days and then allowed to select for protein (PRO) and carbohydrate (CHO) intake. While total food intake was unchanged, rats fed the SBO diet selected lower PRO (3.1 +/- 0.6 vs. 4.9 +/- 0.6 g/day, SBO vs. lard, respectively) and higher CHO (9.6 +/- 0.7 vs. 7.8 +/- 1.2) intakes than those consuming the lard based diet. Comparable differences were seen in a second trial. Current evidence suggests that the regulation of PRO and CHO intake is under serotonergic control. Therefore to determine whether dietary fat is mediating its effect on macronutrient selection via alterations in serotonin (5HT) metabolism, brain stem concentrations of 5HT and its metabolite 5-hydroxyindole acetic acid (5HIAA) and whole brain (minus brain stem) mitochondrial monoamine oxidase (MAO) activity were measured in a separate set of animals fed the SBO or lard diets for 28 days. Vmax of MAO was decreased in rats fed the SBO diets (20.2 +/- 7.4 vs. 27.9 +/- 8.9 nmol/mg prot/20') compared to those fed the lard diets. Km was unaltered by dietary fat fed. The change in activity of MAO was insufficient to alter steady-state levels of 5HT or 5HIAA. We propose that changes in neuronal functioning, induced by altered dietary fat, contributed to the differences seen in PRO and CHO selection. PMID:2441419

  3. Electrochemical activity of glucose oxidase on a poly(ionic liquid) - Au nanoparticle composite.

    SciTech Connect

    Lee, S.; Ringstrand, B. S.; Stone, D. A.; Firestone, M. A.

    2012-01-01

    Glucose oxidase (GOx) adsorbed on an ionic liquid-derived polymer containing internally organized columns of Au nanoparticles exhibits direct electron transfer and bioelectrocatalytic properties towards the oxidation of glucose. The cationic poly(ionic liquid) provides an ideal substrate for the electrostatic immobilization of GOx. The encapsulated Au nanoparticles serve to both promote the direct electron transfer with the recessed enzyme redox centers and impart electronic conduction to the composite, allowing it to function as an electrode for electrochemical detection.

  4. Active Site and Loop 4 Movements with Human Glycolate Oxidase: Implications for Substrate Specificity and Drug Design

    SciTech Connect

    Murray,M.; Holmes, R.; Lowther, W.

    2008-01-01

    Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1, 2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most {alpha}-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with a-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.

  5. Thiophene-degrading Escherichia coli mutants possess sulfone oxidase activity and show altered resistance to sulfur-containing antibiotics.

    PubMed Central

    Juhl, M J; Clark, D P

    1990-01-01

    We have previously isolated mutants of Escherichia coli which show increased oxidation of heterocyclic furan and thiophene substrates. We have now found that strains carrying the thdA mutation express a novel enzyme activity which oxidizes a variety of substrates containing a sulfone (SO2) moiety. Both heterocyclic sulfones (e.g., tetramethylene sulfone) and simple aliphatic sulfones (e.g., ethyl sulfone) were oxidized. The thdA mutants were more resistant than wild-type strains to aromatic sulfone antibiotics such as dapsone. In contrast they showed increased susceptibility to thiolutin, a cyclic antibiotic containing sulfur at the sulfide level of oxidation. Several new thdA mutant alleles were isolated by selecting for increased oxidation of various aliphatic sulfur compounds. These new thdA mutants showed similar sulfone oxidase activity and the same map location (at 10.7 min) as the original thdA1 mutation. The constitutive fadR mutation was required for the phenotypic expression of thdA-mediated oxidation of sulfur compounds. However, the thdA-directed expression of sulfone oxidase activity was not fadR dependent. The thdC and thdD mutations probably protect against the toxicity of thiophene derivatives rather than conferring improved metabolic capability. PMID:2285321

  6. Expanding the laccase-toolbox: a laccase from Corynebacterium glutamicum with phenol coupling and cuprous oxidase activity.

    PubMed

    Ricklefs, Esther; Winkler, Nadine; Koschorreck, Katja; Urlacher, Vlada B

    2014-12-10

    Laccases are oxidases with potential for application in biotechnology. Up to now only fungal laccases have been applied in technical processes, although bacterial laccases are generally easier to handle and more stable at alkaline pH values and elevated temperatures. To increase the toolbox of bacterial laccases and to broaden our knowledge about them, new enzymes have to be characterized. Within this study, we describe the new bacterial laccase CgL1 from Corynebacterium glutamicum. CgL1 was found to oxidize typical laccase substrates like 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), syringaldazine and 2,6-dimethoxyphenol. The enzyme also demonstrates cuprous oxidase activity. Furthermore, CgL1 is active for several hours at temperatures up to 60°C and at alkaline pH, as well as stable in different organic solvents. This makes CgL1 a potential candidate for technical applications. In addition, CgL1 was found to catalyze the CC/CO coupling of several phenolic compounds which can serve as precursors for the synthesis of natural products like antibiotics and phytohormones. This activity and product distribution were influenced by pH value and mediators used. PMID:24910971

  7. Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

    PubMed Central

    Pazmandi, Kitti; Kumar, Brahma V.; Szabo, Krisztina; Boldogh, Istvan; Szoor, Arpad; Vereb, Gyorgy; Veres, Agota; Lanyi, Arpad; Rajnavolgyi, Eva; Bacsi, Attila

    2012-01-01

    Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-?, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3+ pan-T cells resulted in increased secretion of IFN-? and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins. PMID:23251688

  8. Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay.

    PubMed Central

    Missiaen, L; Wuytack, F; Casteels, R

    1988-01-01

    The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5') pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2833248

  9. D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

    PubMed

    Popiolek, Michael; Ross, John F; Charych, Erik; Chanda, Pranab; Gundelfinger, Eckart D; Moss, Stephen J; Brandon, Nicholas J; Pausch, Mark H

    2011-08-19

    Schizophrenia is a highly heritable neuropsychiatric disorder affecting ?1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders. PMID:21700703

  10. Kinetic evidence that methionine sulfoxide reductase A can reveal its oxidase activity in the presence of thioredoxin.

    PubMed

    Kriznik, Alexandre; Boschi-Muller, Sandrine; Branlant, Guy

    2014-04-15

    The mouse methionine sulfoxide reductase A (MsrA) belongs to the subclass of MsrAs with one catalytic and two recycling Cys corresponding to Cys51, Cys198 and Cys206 in Escherichia coli MsrA, respectively. It was previously shown that in the absence of thioredoxin, the mouse and the E. coli MsrAs, which reduce two mol of methionine-O substrate per mol of enzyme, displays an in vitro S-stereospecific methionine oxidase activity. In the present study carried out with E. coli MsrA, kinetic evidence are presented which show that formation of the second mol of Ac-L-Met-NHMe is rate-limiting in the absence of thioredoxin. In the presence of thioredoxin, the overall rate-limiting step is associated with the thioredoxin-recycling process. Kinetic arguments are presented which support the accumulation of the E. coli MsrA under Cys51 sulfenic acid state in the presence of Trx. Thus, the methionine oxidase activity could be operative in vivo without the action of a regulatory protein in order to block the action of Trx as previously proposed. PMID:24632144

  11. Extraction of rice bran extract and some factors affecting its inhibition of polyphenol oxidase activity and browning in potato.

    PubMed

    Boonsiripiphat, Kunnikar; Theerakulkait, Chockchai

    2009-01-01

    The extraction conditions of rice bran extract (RBE), including extraction ratio, extraction time, and extraction temperature, were studied in relation to enzymatic browning inhibition in potato. The inhibitory effect of RBE on potato polyphenol oxidase (PPO) activity and its total phenolic compound content were highest at an extraction ratio of 1:3 (rice bran:water, w/v), extraction time of 30 min, and extraction temperature of 40 degrees C. RBE showed the most inhibitory effect on PPO activity at pH 6.5. However, the inhibitory effect of RBE on potato PPO activity and its total phenolic compound content were decreased at the higher temperature and longer time. PMID:19291577

  12. Synthesis, crystal structures, fluorescence and xanthine oxidase inhibitory activity of pyrazole-based 1,3,4-oxadiazole derivatives

    NASA Astrophysics Data System (ADS)

    Qi, De-Qiang; Yu, Chuan-Ming; You, Jin-Zong; Yang, Guang-Hui; Wang, Xue-Jie; Zhang, Yi-Ping

    2015-11-01

    A series of pyrazole-based 1,3,4-oxadiazole derivatives were rationally designed and synthesized in good yields by following a convenient route. All the newly synthesized molecules were fully characterized by IR, 1H NMR and elemental analysis. Eight compounds were structurally determined by single crystal X-ray diffraction analysis. The fluorescence properties of all the compounds were investigated in dimethyl sulfoxide media. In addition, these newly synthesized compounds were evaluated for in vitro inhibitory activity against commercial enzyme xanthine oxidase (XO) by measuring the formation of uric acid from xanthine. Among the compounds synthesized and tested, 3d and 3e were found to be moderate inhibitory activity against commercial XO with IC50 = 72.4 ?M and 75.6 ?M. The studies gave a new insight in further optimization of pyrazole-based 1,3,4-oxadiazole derivatives with excellent fluorescence properties and XO inhibitory activity.

  13. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. PMID:24530799

  14. Anti-convulsant activity of diazepam and clonidine on metaldehyde-induced seizures in mice: effects on brain gamma-amino butyric acid concentrations and monoamine oxidase activity.

    PubMed

    Homeida, A M; Cooke, R G

    1982-09-01

    Metaldehyde when administered orally to mice at a dose of 1 g kg-1 produced convulsions and death within 2 h. Brain concentrations of gamma-aminobutyric acid (GABA) were significantly reduced and monoamine oxidase (MAO) activity significantly increased in these animals relative to controls. Treatment with either intraperitoneal diazepam or clonidine 20 min after administration of metaldehyde delayed the onset of toxic symptoms and reduced the mortality rate. In those mice which survived longer than 5 h, brain concentrations of GABA, though still not restored to control values, were significantly higher than those in the mice which died. Clonidine, unlike diazepam, also inhibited the increase in brain MAO activity. PMID:7143555

  15. Cardiac Cytochrome c Oxidase Activity and Contents of Submits 1 and 4 are Altered in Offspring by Low Prenatal Intake by Rat Dams

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been reported previously that the offspring of rat dams consuming low dietary copper (Cu) during pregnancy and lactation experience a deficiency in cardiac cytochrome c oxidase (CCO) characterized by reduced catalytic activity and mitochondrial- and nuclear-subunit content after postnatal day...

  16. NAD kinase levels control the NADPH concentration in human cells.

    PubMed

    Pollak, Nadine; Niere, Marc; Ziegler, Mathias

    2007-11-16

    NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H(2)O(2). Overexpression of NADK resulted in a 4-5-fold increase in the NADPH, but not NADP(+), concentration, although the recombinant enzyme phosphorylated preferentially NAD(+). Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP(+)), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress. PMID:17855339

  17. Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression.

    PubMed

    Ye, Mao; Li, Gang; Liang, Wei Qu; Liu, Yu Huan

    2010-07-01

    Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55 degrees C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0-10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > alpha-naphthol > guaiacol > SGZ > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing. PMID:20358193

  18. Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

    PubMed

    Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A

    2015-12-01

    Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD. PMID:25398695

  19. Relationship between NADPH and Th1/Th2 ratio in patients with non-Hodgkin lymphoma who have been exposed to pesticides

    PubMed Central

    Zahzeh, Meriem Rabia; Loukidi, Bouchra; Meziane, Warda; Haddouche, Mustapha; Mesli, Naima; Zouaoui, Zahia; Aribi, Mourad

    2015-01-01

    The effect of pesticides on nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), including its level and relationship with the T helper 1 (Th1)/Th2 ratio, in patients suffering from non-Hodgkin lymphoma (NHL) was investigated. One hundred newly diagnosed patients with aggressive NHL (53 men, 47 women) and 40 healthy age-, sex-, and body mass index-matched controls (23 men, 17 women), exposed or not to pesticides, were recruited for a cross-sectional study conducted at the Clinical Hematology Departments of Tlemcen and Sidi Bel-Abbès University Medical Centers in the northwest of Algeria. NADPH levels were significantly increased in patients compared with controls; and in exposed patients compared with those not exposed, and controls (one-way analysis of variance; P=0.000). Albumin, glutathione peroxidase, superoxide dismutase, catalase activity, and oxygen radical absorbance capacity levels were significantly decreased in patients compared with in the control group. Oxygen radical absorbance capacity levels were significantly decreased in exposed patients compared with in unexposed patients; however, malondialdehyde levels were significantly increased in exposed patients when compared with controls and unexposed patients. Protein carbonyl and xanthine oxidase levels were significantly increased in exposed patients compared with controls; meanwhile, there were no significant differences between the two patient groups or between unexposed patients and controls. The Th1/Th2 ratio was significantly decreased in patients when compared with controls; the neutrophil-to-lymphocyte ratio was significantly increased (for both comparisons, P<0.001). In addition, NADPH was strongly associated with NHL (Mantel–Haenszel common odds ratio estimate =5.55; 95% confidence interval, 2.22–13.88; P=0.000). Moreover, NADPH levels were significantly negatively related to the Th1/Th2 ratio, either in exposed patients or in unexposed patients (respectively, r=?0.498 [P=0.004] and r=?0.327 [P=0.006]). In conclusion, pesticide exposure was strongly associated with NADPH alteration in NHL. The relationship between NADPH and Th1/Th2 ratio should focus on new therapeutic strategies for the disease. PMID:25878515

  20. Effects of aqueous soybean, mistletoe and red clover extracts on activities of adenosine deaminase and xanthine oxidase enzyme.

    PubMed

    Namuslu, M; Kocaoglu, H; Celik, H T; Avci, A; Devrim, E; Genc, Y; Gocmen, E; Erguder, I B; Durak, I

    2014-01-01

    Soybean (Glycine max), mistletoe (Viscum album) and red clover (Trifolium pratence) have been argued to have anti-cancer effects. In the present study it was aimed to investigate possible effects of these plant extracts on the activities of DNA turn-over enzymes, namely adenosine deaminase (ADA) and xanthine oxidase (XO) in cancerous and non-cancerous gastric and colon tissues. For this aim, 6 cancerous and 6 non-cancerous adjacent human gastric tissues, and 7 cancerous and 7 non-cancerous adjacent colon tissues were obtained by surgical operations. Our results suggest that aqueous soybean, mistletoe and red clover extracts may exhibit anti-tumoral activity by depleting hypoxanthine concentration in the cancer cells through XO activation, which may lead to lowered salvage pathway activity necessary for the cancer cells to proliferate in the cancerous colon tissue. Some foods like soybean, mistletoe and red clover may provide nutritional support to medical cancer therapy through inhibiting and/or activating key enzymes in cancer metabolism (Tab. 4, Ref.?33). PMID:25023428

  1. Systematic investigation of changes in oxidized cerebral cytochrome c oxidase concentration during frontal lobe activation in healthy adults

    PubMed Central

    Kolyva, Christina; Tachtsidis, Ilias; Ghosh, Arnab; Moroz, Tracy; Cooper, Chris E.; Smith, Martin; Elwell, Clare E.

    2012-01-01

    Using transcranial near-infrared spectroscopy (NIRS) to measure changes in the redox state of cerebral cytochrome c oxidase (?[oxCCO]) during functional activation in healthy adults is hampered by instrumentation and algorithm issues. This study reports the ?[oxCCO] response measured in such a setting and investigates possible confounders of this measurement. Continuous frontal lobe NIRS measurements were collected from 11 healthy volunteers during a 6-minute anagram-solving task, using a hybrid optical spectrometer (pHOS) that combines multi-distance frequency and broadband components. Only data sets showing a hemodynamic response consistent with functional activation were interrogated for a ?[oxCCO] response. Simultaneous systemic monitoring data were also available. Possible influences on the ?[oxCCO] response were systematically investigated and there was no effect of: 1) wavelength range chosen for fitting the measured attenuation spectra; 2) constant or measured, with the pHOS in real-time, differential pathlength factor; 3) systemic hemodynamic changes during functional activation; 4) changes in optical scattering during functional activation. The ?[oxCCO] response measured in the presence of functional activation was heterogeneous, with the majority of subjects showing significant increase in oxidation, but others having a decrease. We conclude that the heterogeneity in the ?[oxCCO] response is physiological and not induced by confounding factors in the measurements. PMID:23082295

  2. Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

    PubMed

    Maia, Luisa B; Pereira, Vânia; Mira, Lurdes; Moura, José J G

    2015-01-27

    Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 ?M) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised. PMID:25537183

  3. CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice

    PubMed Central

    Magome, Hiroshi; Nomura, Takahito; Hanada, Atsushi; Takeda-Kamiya, Noriko; Ohnishi, Toshiyuki; Shinma, Yuko; Katsumata, Takumi; Kawaide, Hiroshi; Kamiya, Yuji; Yamaguchi, Shinjiro

    2013-01-01

    Bioactive gibberellins (GAs) control many aspects of growth and development in plants. GA1 has been the most frequently found bioactive GA in various tissues of flowering plants, but the enzymes responsible for GA1 biosynthesis have not been fully elucidated due to the enzymes catalyzing the 13-hydroxylation step not being identified. Because of the lack of mutants defective in this enzyme, biological significance of GA 13-hydroxylation has been unknown. Here, we report that two cytochrome P450 genes, CYP714B1 and CYP714B2, encode GA 13-oxidase in rice. Transgenic Arabidopsis plants that overexpress CYP714B1 or CYP714B2 show semidwarfism. There was a trend that the levels of 13-OH GAs including GA1 were increased in these transgenic plants. Functional analysis using yeast or insect cells shows that recombinant CYP714B1 and CYP714B2 proteins can convert GA12 into GA53 (13-OH GA12) in vitro. Moreover, the levels of 13-OH GAs including GA1 were decreased, whereas those of 13-H GAs including GA4 (which is more active than GA1) were increased, in the rice cyp714b1 cyp714b2 double mutant. These results indicate that CYP714B1 and CYP714B2 play a predominant role in GA 13-hydroxylation in rice. The double mutant plants appear phenotypically normal until heading, but show elongated uppermost internode at the heading stage. Moreover, CYP714B1 and CYP714B2 expression was up-regulated by exogenous application of bioactive GAs. Our results suggest that GA 13-oxidases play a role in fine-tuning plant growth by decreasing GA bioactivity in rice and that they also participate in GA homeostasis. PMID:23319637

  4. Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

    PubMed Central

    Mock, Hans-Peter; Keetman, Ulrich; Kruse, Elisabeth; Rank, Barbara; Grimm, Bernhard

    1998-01-01

    We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

  5. Antimicrobial activity and hydrophobicity of edible whey protein isolate films formulated with nisin and/or glucose oxidase.

    PubMed

    Murillo-Martínez, María M; Tello-Solís, Salvador R; García-Sánchez, Miguel A; Ponce-Alquicira, Edith

    2013-04-01

    The use of edible antimicrobial films has been reported as a means to improve food shelf life through gradual releasing of antimicrobial compounds on the food surface. This work reports the study on the incorporation of 2 antimicrobial agents, nisin (N), and/or glucose oxidase (GO), into the matrix of Whey protein isolate (WPI) films at pH 5.5 and 8.5. The antimicrobial activity of the edible films was evaluated against Listeria innocua (ATCC 33090), Brochothrix thermosphacta (NCIB10018), Escherichia coli (JMP101), and Enterococcus faecalis (MXVK22). In addition, the antimicrobial activity was related to the hydrophobicity and water solubility of the WPI films. The greatest antibacterial activity was observed in WPI films containing only GO. The combined addition of N and GO resulted in films with lower antimicrobial activity than films with N or GO alone. In most cases, a pH effect was observed as greater antimicrobial response at pH 5.5 as well as higher film matrix hydrophobicity. WPI films supplemented with GO can be used in coating systems suitable for food preservation. PMID:23488765

  6. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2015-01-01

    Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6?kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9?kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ?1.65?Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50?Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI). PMID:26057806

  7. Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants.

    PubMed

    Broothaerts, W; McPherson, J; Li, B; Randall, E; Lane, W D; Wiersma, P A

    2000-12-01

    A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed. PMID:11141262

  8. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

    PubMed Central

    Kanade, Santosh R.; Paul, Beena; Rao, A. G. Appu; Gowda, Lalitha R.

    2006-01-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  9. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.

    PubMed

    Kanade, Santosh R; Paul, Beena; Rao, A G Appu; Gowda, Lalitha R

    2006-05-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  10. The effect of ultrasound on particle size, color, viscosity and polyphenol oxidase activity of diluted avocado puree.

    PubMed

    Bi, Xiufang; Hemar, Yacine; Balaban, Murat O; Liao, Xiaojun

    2015-11-01

    The effect of ultrasound treatment on particle size, color, viscosity, polyphenol oxidase (PPO) activity and microstructure in diluted avocado puree was investigated. The treatments were carried out at 20 kHz (375 W/cm(2)) for 0-10 min. The surface mean diameter (D[3,2]) was reduced to 13.44 ?m from an original value of 52.31 ?m by ultrasound after 1 min. A higher L(?) value, ?E value and lower a(?) value was observed in ultrasound treated samples. The avocado puree dilution followed pseudoplastic flow behavior, and the viscosity of diluted avocado puree (at 100 s(-1)) after ultrasound treatment for 1 min was 6.0 and 74.4 times higher than the control samples for dilution levels of 1:2 and 1:9, respectively. PPO activity greatly increased under all treatment conditions. A maximum increase of 25.1%, 36.9% and 187.8% in PPO activity was found in samples with dilution ratios of 1:2, 1:5 and 1:9, respectively. The increase in viscosity and measured PPO activity might be related to the decrease in particle size. The microscopy images further confirmed that ultrasound treatment induced disruption of avocado puree structure. PMID:25899308

  11. Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

    PubMed

    Graz, Marcin; Jarosz-Wilko?azka, Anna; Pawlikowska-Pawlega, Bozena

    2009-06-01

    The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu(2)O, Al(2)O(3), ZnO, CuFe(2)O(4)Zn, CdO, and MnO(2)) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu(2)O- and ZnO-amended plates was observed. ZnO, CdO and Cu(2)O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu(2)O, MnO(2), ZnO and CuFe(2)O(4)Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu(2)O, ZnO and MnO(2). Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number. PMID:18985279

  12. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells.

    PubMed

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E; Magnusson, S Peter; Kjaer, Michael

    2015-06-26

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used ?-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  13. Histochemical detection of monoamine oxidase activity in smooth muscle and epithelial tissues of Mytilus edulis L. and Mytilus galloprovincialis Lmk.

    PubMed

    Gilloteaux, J

    1979-01-01

    Monoamine oxidase (MAO) activity was detected in the glio-interstitial tissue of the anterior byssus retractor muscle (ABRM) of Mytilus by the use of either dopamine or 5-HT as substrate and nitro blue tetrazolium salt as an electron receptor. Both substrates gave superimposable patterns. However, dopamine reactivity was very weak. Discrete but clear MAO activity was detected in the periphery of the smooth muscle cell, i.e., at the level of the peripherally localized mitochondria. The presence of MAO in the epithelium of the buccal palps was also demonstrated. These histochemical observations can be correlated with certain ultrastructural aspects, in particular the presence electron dense organelles profiles in glio-interstitial cells. This MAO activity is discussed in relationship with the high 5-HT content of ABRM and the control of its relaxation. Pigment formation and/or collagen tanning are discussed in terms of a possible relationship with inactivation of biogenic amines. These results complement previous data concerning the glio-interstitial tissue found in close association with muscle and nerve structures. PMID:118621

  14. Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance.

    PubMed Central

    Brass, C A; Narciso, J; Gollan, J L

    1991-01-01

    It has been widely proposed that conversion of xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD), underlies ischemic/reperfusion injury, although the relationship of this conversion to hypoxia and its physiologic control have not been defined. This study details the time course and control of this enzymatic interconversion. In a functionally intact, isolated perfused rat liver model, mean % XOD activity increased as a function of both the duration (25 to 45% in 3 h) and degree (r = 0.97) of hypoxia. This process was markedly accelerated in ischemic liver by an overnight fast (45 vs. 30% at 2 h), and by imposing a short period of in vivo ischemia (cardiopulmonary arrest 72%). Moreover, only under these conditions was there a significant rise in the XOD activity due to the conformationally altered XDH molecule (XODc, 18%), as well as concomitant morphologic injury. Neither circulating white blood cells nor thrombosis appeared to contribute to the effects of in vivo ischemia on enzyme conversion. Thus, it is apparent that conversion to the free radical-producing state, with high levels of XOD activity and concurrent cellular injury, can be achieved during a relatively short period of hypoxia under certain well-defined physiologic conditions, in a time course consistent with its purported role in modulating reperfusion injury. These data also suggest that the premorbid condition of organ donors (e.g., nutritional status and relative state of hypoxia) is important in achieving optimal organ preservation. Images PMID:1991828

  15. Identification of the NAD(P)H binding site of eukaryotic UDP-galactopyranose mutase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Solano, Luis M; Oppenheimer, Michelle; Robinson, Reeder M; Ellerbrock, Jacob F; Sobrado, Pablo; Tanner, John J

    2012-10-31

    UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery. PMID:23036087

  16. Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

    PubMed Central

    Woodman, R C; Ruedi, J M; Jesaitis, A J; Okamura, N; Quinn, M T; Smith, R M; Curnutte, J T; Babior, B M

    1991-01-01

    Resting and phorbol-activated human neutrophils were separated by treatment with Triton X-100 into detergent-extractable and cytoskeleton fractions. Respiratory burst oxidase activity was restricted entirely to the cytoskeleton. The cytoskeleton also contained approximately 15% of the neutrophil cytochrome b558, an oxidase-associated heme protein, as well as most of the oxidase-related cytosolic polypeptide p67phox. In contrast, the components of the oxidase-associated phosphoprotein family p47phox were found almost exclusively in the detergent extract, suggesting that p47phox is needed for oxidase activation but not for O2- production by the activated oxidase. Activation of the oxidase had no apparent effect on the distribution of any of these species between the cytoskeleton and the detergent extract. Our results support earlier studies implying that the cytoskeleton participates in an important way in regulating the activity of the O2(-)-forming respiratory burst oxidase of neutrophils. Images PMID:1849148

  17. The fronto-parietal cortex of the prosimian Galago: patterns of cytochrome oxidase activity and motor maps.

    PubMed

    Fogassi, L; Gallese, V; Gentilucci, M; Luppino, G; Matelli, M; Rizzolatti, G

    1994-01-31

    We mapped the motor areas of the prosimian Galago crassicaudatus using intracortical electrical microstimulation and morphological and histochemical (cytochrome oxidase) techniques. Stimulation data showed that on the brain convexity there is an area (area Frontalis posterior, F post.) from which movements could be evoked at low threshold (< 10 microA). This area is somatotopically organized, with the leg represented medially, the arm centrally and the face and mouth laterally. Proximal and distal movements are not segregated. Most of the evoked movements, even at threshold, consist of movements involving two or more joints. F post. is characterized by a three-band cytochrome oxidase activity pattern. It has an agranular structure, but it lacks pyramidal cells that are larger than those observed in other areas. In front of F post. there is an area histochemically similar to it, Frontalis intermedialis (F int.). This area consists of two cytoarchitectonic divisions: an agranular division (F int. pars caudalis) and a disgranular division (F int. pars rostralis). The excitability threshold of F int. is relatively high (10 to 30 microA). Eye, ear and neck movements are elicited from its lateral part, whereas trunk movements associated with limb movements are elicited from its medial part. Caudal to F post., there is another region from which movements can be evoked with currents between 10 to 30 microA. This region has the same medio-lateral somatotopic arrangement of F post. Typically, single joint movements are elicited from it. Proximal and distal movements are not segregated. In spite of its homogeneity in terms of motor response, the posterior excitable region is formed by two anatomically separate areas: anterior somatic area (S ant.) and posterior somatic area (S post.). S ant. has a typical koniocortex structure, whereas S post, resembles the parakoniocortex as defined by Sanides (J. Hirnforsch., 9 (1967) 225-252). Histochemically both areas are made up of four longitudinal stripes differing for enzymatic activity. The three superficial stripes tend to merge together and are sharply separated from a deeply located, light stripe. This stripe is homogeneous in S ant., whilst its central part shows an increase in activity in S post. The possible homologies between the motor and somatic areas of the galago and monkey as well as their role in movement control are discussed. PMID:8185856

  18. Diversity and relationships in key traits for functional and apparent quality in a collection of eggplant: fruit phenolics content, antioxidant activity, polyphenol oxidase activity, and browning.

    PubMed

    Plazas, Mariola; López-Gresa, María P; Vilanova, Santiago; Torres, Cristina; Hurtado, Maria; Gramazio, Pietro; Andújar, Isabel; Herráiz, Francisco J; Bellés, José M; Prohens, Jaime

    2013-09-18

    Eggplant (Solanum melongena) varieties with increased levels of phenolics in the fruit present enhanced functional quality, but may display greater fruit flesh browning. We evaluated 18 eggplant accessions for fruit total phenolics content, chlorogenic acid content, DPPH scavenging activity, polyphenol oxidase (PPO) activity, liquid extract browning, and fruit flesh browning. For all the traits we found a high diversity, with differences among accessions of up to 3.36-fold for fruit flesh browning. Variation in total content in phenolics and in chlorogenic acid content accounted only for 18.9% and 6.0% in the variation in fruit flesh browning, and PPO activity was not significantly correlated with fruit flesh browning. Liquid extract browning was highly correlated with chlorogenic acid content (r = 0.852). Principal components analysis (PCA) identified four groups of accessions with different profiles for the traits studied. Results suggest that it is possible to develop new eggplant varieties with improved functional and apparent quality. PMID:23972229

  19. Fusion of a Xylan-Binding Module to Gluco-Oligosaccharide Oxidase Increases Activity and Promotes Stable Immobilization

    PubMed Central

    Vuong, Thu V.; Master, Emma R.

    2014-01-01

    The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides. PMID:24736604

  20. Protonation of the binuclear active site in cytochrome c oxidase decreases the reduction potential of CuB.

    PubMed

    Blomberg, Margareta R A; Siegbahn, Per E M

    2015-10-01

    One of the remaining mysteries regarding the respiratory enzyme cytochrome c oxidase is how proton pumping can occur in all reduction steps in spite of the low reduction potentials observed in equilibrium titration experiments for two of the active site cofactors, CuB(II) and Fea3(III). It has been speculated that, at least the copper cofactor can acquire two different states, one metastable activated state occurring during enzyme turnover, and one relaxed state with lower energy, reached only when the supply of electrons stops. The activated state should have a transiently increased CuB(II) reduction potential, allowing proton pumping. The relaxed state should have a lower reduction potential, as measured in the titration experiments. However, the structures of these two states are not known. Quantum mechanical calculations show that the proton coupled reduction potential for CuB is inherently high in the active site as it appears after reaction with oxygen, which explains the observed proton pumping. It is suggested here that, when the flow of electrons ceases, a relaxed resting state is formed by the uptake of one extra proton, on top of the charge compensating protons delivered in each reduction step. The extra proton in the active site decreases the proton coupled reduction potential for CuB by almost half a volt, leading to agreement with titration experiments. Furthermore, the structure for the resting state with an extra proton is found to have a hydroxo-bridge between CuB(II) and Fea3(III), yielding a magnetic coupling that can explain the experimentally observed EPR silence. PMID:26072193

  1. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    SciTech Connect

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ?1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  2. Polyphenol oxidase activity and differential accumulation of polyphenolics in seed coats of pinto bean (Phaseolus vulgaris L.) characterize postharvest color changes.

    PubMed

    Marles, M A Susan; Vandenberg, Albert; Bett, Kirstin E

    2008-08-27

    Postharvest darkening of pinto bean (Phaseolus vulgaris L.) was evaluated in a population of recombinant inbred lines derived from a cross between CDC Pintium (a regular-darkening line) and 1533-15 (a slow-darkening line). Flavonoid metabolite concentrations, polyphenol oxidase activity, lignin concentration, and seed coat anatomy characteristics were assessed for cosegregation with the darkening phenotype. Significantly lower kaempferol concentrations (p = 0.00001) together with differences in polyphenol oxidase activity (p = 0.0045) were two of the key findings associated with these recombinant inbred lines. In addition, two different assays (thioglycolic acid and Klason lignin) to quantify lignin together with an assessment of extractable condensed tannin were used to estimate the contribution of these polymers to changes in the seed coat tissue. This is the first report of precise biochemical characterization of polyphenolics that associate with postharvest darkening in legumes. PMID:18666779

  3. Associations between platelet monoamine oxidase-B activity and acquired colour vision loss in a fish-eating population.

    PubMed

    Stamler, Christopher John; Mergler, Donna; Abdelouahab, Nadia; Vanier, Claire; Chan, Hing Man

    2006-01-01

    Platelet monoamine oxidase-B (MAO-B) has been considered a surrogate biochemical marker of neurotoxicity, as it may reflect changes in the monoaminergic system in the brain. Colour vision discrimination, in part a dopamine dependent process, has been used to identify early neurological effects of some environmental and industrial neurotoxicants. The objective of this cross-sectional study was to explore the relationship between platelet MAO-B activity and acquired colour discrimination capacity in fish-consumers from the St. Lawrence River region of Canada. Assessment of acquired dyschromatopsia was determined using the Lanthony D-15 desaturated panel test. Participants classified with dyschromatopsia (n=81) had significantly lower MAO-B activity when compared to those with normal colour vision (n=32) (26.5+/-9.6 versus 31.0+/-9.9 nmol/min/20 microg, P=0.030)). Similarly, Bowman's Colour Confusion Index (CCI) was inversely correlated with MAO-B activity when the vision test was performed with the worst eye only (r=-0.245, P=0.009), the best eye only (r=-0.188, P=0.048) and with both eyes together (r=-0.309, P=0.001). Associations remained significant after adjustment for age and gender when both eyes (P=0.003) and the worst eye (P=0.045) were tested. Adjustment for heavy smoking weakened the association between MAO-B and CCI in the worst eye (P=0.140), but did not alter this association for both eyes (P=0.006). Adjustment for blood-mercury concentrations did not change the association. This study suggests a relationship between reduced MAO-B activity and acquired colour vision loss and both are associated with tobacco smoking. Therefore, results show that platelet MAO-B may be used as a surrogate biochemical marker of acquired colour vision loss. PMID:16806814

  4. Catecholamines oxidation by xanthine oxidase.

    PubMed

    Foppoli, C; Coccia, R; Cini, C; Rosei, M A

    1997-03-15

    Dopamine and structurally related catecholamines in the presence of hydrogen peroxide are oxidized in vitro by xanthine oxidase producing the corresponding melanin pigments. The kinetic parameters of the reaction, measured as aminochrome formation, have been calculated. The rate of peroxidation depends on enzyme and hydrogen peroxide concentration. The optimum pH for the peroxidative activity of the enzyme is around 8.5. Activation of the peroxidative reaction is also elicited by catechol compounds through a redox cycle mechanism. Implications about the possible biochemical relevance of xanthine oxidase activity on catecholamines oxidation are discussed. PMID:9101714

  5. Enhanced Expression and Activation of the Alternative Oxidase during Infection of Arabidopsis with Pseudomonas syringae pv tomato1

    PubMed Central

    Simons, Bert H.; Millenaar, Frank F.; Mulder, Lonneke; Van Loon, Leendert C.; Lambers, Hans

    1999-01-01

    Cyanide-resistant (“alternative”) respiration was studied in Arabidopsis during incompatible and compatible infection with Pseudomonas syringae pv tomato DC3000. Total leaf respiration increased as the leaves became necrotic, as did the cyanide-resistant component that was sensitive to salicylhydroxamic acid. Infiltration of leaves with an avirulent strain rapidly induced alternative oxidase (AOX) mRNA, whereas the increase was delayed in the compatible combination. The increase in mRNA correlated with the increase in AOX protein. Increased expression was confined to the infected leaves, in contrast to the pathogenesis-related protein-1, which was induced systemically. Virtually all of the AOX protein was in the reduced (high-activity) form. Using transgenic NahG and mutant npr1-1 and etr1-1 plants, we established that the rapid induction of the AOX was associated with necrosis and that ethylene, but not salicylic acid, was required for its induction. Increased pyruvate levels in the infected leaves suggested that increased substrate levels were respired through the alternative pathway; however, in the control leaves and the infected leaves, respiration was not inhibited by salicylhydroxamic acid alone. Increased respiration appeared to be associated primarily with symptom expression rather than resistance reactions. PMID:10364404

  6. Effects of gamma irradiation on the radiation-resistant bacteria and polyphenol oxidase activity in fresh kale juice

    NASA Astrophysics Data System (ADS)

    Kim, Dongho; Song, Hyunpa; Lim, Sangyong; Yun, Hyejeong; Chung, Jinwoo

    2007-07-01

    Gamma radiation was performed to prolong the shelf life of natural kale juice. The total aerobic bacteria in fresh kale juice, prepared by a general kitchen process, was detected in the range of 10 6 cfu/ml, and about 10 2 cfu/ml of the bacteria survived in the juice in spite of gamma irradiation treatment with a dose of 5 kGy. Two typical radiation-resistant bacteria, Bacillus megaterium and Exiguobacterium acetylicum were isolated and identified from the 5 kGy-irradiated kale juices. The D10 values of the vegetative cell and endospore of the B. megaterium in peptone water were 0.63±0.05 and 1.52±0.05 kGy, respectively. The D10 value of the E. acetylicum was calculated as 0.65±0.06 kGy. In the inoculation test, the growth of the surviving B. megaterium and E. acetylicum in the 3-5 kGy-irradiated kale juice retarded and/or decreased significantly during a 3 d post-irradiation storage period. However, there were no significant differences in the residual polyphenol oxidase activity and browning index between the nonirradiated control and the gamma irradiated kale juice during a post-irradiation period.

  7. Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.

    PubMed

    Güllçin, Ilhami; Küfrevio?lu, O Irfan; Oktay, Münir

    2005-06-01

    Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30 degrees C respectively and Km and Vmax values were 7.90 x 10(-4) M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, beta-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 x 10(-9)M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis. PMID:16119202

  8. Cytokinin Oxidase from Wheat

    PubMed Central

    Laloue, Michel; Fox, J. Eugene

    1989-01-01

    As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N6-(?2-isopentenyl)adenosine to adenosine at a Vmax of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the Km being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N6-(?2-isopentenyl)adenine and zeatin riboside are also degraded, but N6-(?2-isopentenyl)adenosine-5?-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N6-(?2-isopentenyl)adenosine. The degradation of N6-(?2-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N?-phenylurea. PMID:16666895

  9. Comparison of the Inhibition of Monoamine Oxidase and Butyrylcholinesterase Activities by Infusions from Green Tea and Some Citrus Peels

    PubMed Central

    Ademosun, Ayokunle O.

    2014-01-01

    This study sought to investigate the effect of infusions from green tea (Camellia sinensis) and some citrus peels [shaddock (Citrus maxima), grapefruit (Citrus paradisi), and orange (Citrus sinensis)] on key enzymes relevant to the management of neurodegenerative conditions [monoamine oxidase (MAO) and butyrylcholinesterase (BChE)]. The total phenol contents and antioxidant activities as typified by their 2,2?-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals scavenging abilities, ferric reducing antioxidant properties, and Fe2+ chelating abilities were also investigated. Green tea had the highest total phenol (43.3?mg/g) and total flavonoid (16.4?mg/g) contents, when compared to orange [total phenol (19.6?mg/g), total flavonoid (6.5?mg/g)], shaddock [total phenol (16.3?mg/g), total flavonoid (5.2?mg/g)], and grapefruit [total phenol (17.7?mg/g), total flavonoid (5.9?mg/g)]. Orange (EC50 = 1.78?mg/mL) had the highest MAO inhibitory ability, while green tea had the least MAO inhibitory ability (EC50 = 2.56?mg/mL). Similarly, green tea had the least BChE inhibitory ability (EC50 = 5.43?mg/mL) when compared to the citrus peels' infusions. However, green tea infusions had the strongest highest ABTS radical scavenging ability, reducing power, and Fe2+ chelating ability. The inhibition of MAO and BChE activities by the green tea and citrus peels infusions could make them good dietary means for the prevention/management of neurodegenerative conditions. PMID:25243093

  10. RhoA/Rho kinase mediates TGF-?1-induced kidney myofibroblast activation through Poldip2/Nox4-derived reactive oxygen species.

    PubMed

    Manickam, Nagaraj; Patel, Mandakini; Griendling, Kathy K; Gorin, Yves; Barnes, Jeffrey L

    2014-07-15

    The small G proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory have shown that NADPH oxidase Nox4-derived ROS are involved in transforming growth factor (TGF)-?1-induced rat kidney myofibroblast differentiation assessed by the acquisition of an ?-smooth muscle actin (?-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link among Rac1 and RhoA and Nox4-dependent ROS generation in TGF-?1-induced kidney myofibroblast activation. TGF-?1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant ?-SMA and Fn-EIIIA expression. RhoA but not Rac1 was involved in TGF-?1 induction of Nox4 signaling of kidney myofibroblast activation. TGF-?1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering RNA and ROCK using Y-27632 significantly reduced TGF-?1-induced stimulation of Nox4 protein, NADPH oxidase activity, and ?-SMA and Fn-EIIIA expression. Treatment with diphenyleneiodonium, an inhibitor of NADPH oxidase, did not decrease RhoA activation but inhibited TGF-?1-induced ?-SMA and Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase (DNA-directed) ?-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis. PMID:24872317

  11. Natural low-molecular mass organic compounds with oxidase activity as organocatalysts

    PubMed Central

    Nishiyama, Tatsuya; Hashimoto, Yoshiteru; Kusakabe, Hitoshi; Kumano, Takuto; Kobayashi, Michihiko

    2014-01-01

    Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of l-ascorbic acid and l-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H2O2 proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H2O2. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature. PMID:25411318

  12. Ox-LDL Induces Dysfunction of Endothelial Progenitor Cells via Activation of NF-?B

    PubMed Central

    Ji, Kang-ting; Qian, Lu; Nan, Jin-liang; Xue, Yang-jing; Zhang, Su-qin; Wang, Guo-qiang; Yin, Ri-peng; Zhu, Yong-jin; Wang, Lu-ping; Ma, Jun; Liao, Lian-ming; Tang, Ji-fei

    2015-01-01

    Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity. Endothelial progenitor cells (EPCs) are thought to contribute to endothelial recovery after arterial injury. Oxidized low-density lipoprotein (ox-LDL) can induce EPC dysfunction, but the underlying mechanism is not well understood. Human EPCs were cultured in endothelial growth medium supplemented with VEGF (10?ng/mL) and bFGF (10?ng/mL). The cells were treated with ox-LDL (50?µg/mL). EPC proliferation was assayed by using CCK8 kits. Expression and translocation of nuclear factor-kabba B (NF-?B) were evaluated. The level of reactive oxygen species (ROS) in cells was measured using H2DCF-DA as a fluorescence probe. The activity of NADPH oxidase activity was determined by colorimetric assay. Ox-LDL significantly decreased the proliferation, migration, and adhesion capacity of EPCs, while significantly increased ROS production and NADPH oxidase expression. Ox-LDL induced NF-?B P65 mRNA expression and translocation in EPCs. Thus ox-LDL can induce EPC dysfunction at least by increasing expression and translocation of NF-?B P65 and NADPH oxidase activity, which represents a new mechanism of lipidemia-induced vascular injury. PMID:25821786

  13. An Oxidized Extracellular Redox State Increases Nox1 Expression and Proliferation in Vascular Smooth Muscle Cells via EGFR Activation

    PubMed Central

    Stanic, Bojana; Katsuyama, Masato; Miller, Francis J.

    2010-01-01

    Objective Generation of reactive oxygen species (ROS) by Nox-based NADPH oxidases activate redox-dependent signaling pathways and contribute to development of “oxidative stress” in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans, however; cellular mechanisms by which extracellular redox state may cause disease are not known. In this study, we examined the effect of an oxidized extracellular redox state (Eh) on expression of NADPH oxidases in vascular cells. Methods and Results Aortic segments and cultured aortic smooth muscle cells (SMCs) were exposed to Eh between ?150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized Eh increased expression of Nox1 and resulted in Nox1-dependent proliferation of SMCs. Oxidized Eh rapidly induced epidermal growth factor receptor (EGFR) phosphorylation via shedding of EGF-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent phosphorylation of the transcription factors activating transcription factor-1 (ATF-1) and cAMP-response-element-binding protein (CREB). Inhibition of EGFR or ERK1/2 activation, or siRNA to ATF-1, prevented the increase in Nox1 expression. Conclusion Our results identify a novel mechanism whereby extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease. PMID:20814013

  14. X-ray Crystal Structure of Arsenite-Inhibited Xanthine Oxidase:[mu]-Sulfido,[mu]-Oxo Double Bridge between Molybdenum and Arsenic in the Active Site

    SciTech Connect

    Cao, Hongnan; Hall, James; Hille, Russ

    2012-10-23

    Xanthine oxidoreductase is a molybdenum-containing enzyme that catalyzes the hydroxylation reaction of sp{sup 2}-hybridized carbon centers of a variety of substrates, including purines, aldehydes, and other heterocyclic compounds. The complex of arsenite-inhibited xanthine oxidase has been characterized previously by UV-vis, electron paramagnetic resonance, and X-ray absorption spectroscopy (XAS), and the catalytically essential sulfido ligand of the square-pyrimidal molybdenum center has been suggested to be involved in arsenite binding through either a {mu}-sulfido,{mu}-oxo double bridge or a single {mu}-sulfido bridge. However, this is contrary to the crystallographically observed single {mu}-oxo bridge between molybdenum and arsenic in the desulfo form of aldehyde oxidoreductase from Desulfovibrio gigas (an enzyme closely related to xanthine oxidase), whose molybdenum center has an oxo ligand replacing the catalytically essential sulfur, as seen in the functional form of xanthine oxidase. Here we use X-ray crystallography to characterize the molybdenum center of arsenite-inhibited xanthine oxidase and solve the structures of the oxidized and reduced inhibition complexes at 1.82 and 2.11 {angstrom} resolution, respectively. We observe {mu}-sulfido,{mu}-oxo double bridges between molybdenum and arsenic in the active sites of both complexes. Arsenic is four-coordinate with a distorted trigonal-pyramidal geometry in the oxidized complex and three-coordinate with a distorted trigonal-planar geometry in the reduced complex. The doubly bridged binding mode is in agreement with previous XAS data indicating that the catalytically essential sulfur is also essential for the high affinity of reduced xanthine oxidoreductase for arsenite.

  15. Prenatal Cu intake by rat dams is the principle determinant of cardiac cytochrome c oxidase activity in their offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preceding studies have shown that cardiac cytochrome c oxidase (CCO) deficiency occurs in the offspring of Cu-deficient rats on postnatal days (PND) 15 and 21. In order to determine if the CCO deficiency resulted from low prenatal Cu intake rather than from low postnatal Cu intake, pups from dams fe...

  16. Inhibitory effect of microwaved thinned nectarine extracts on polyphenol oxidase activity.

    PubMed

    Redondo, Diego; Venturini, María E; Oria, Rosa; Arias, Esther

    2016-04-15

    By-products from agricultural practices or from the fruit processing industry are a source of bioactive compounds that could be used in the food industry. Such by-products include thinned fruits, which are expected to contain high quantities of interesting compounds. One possible application of this fruits is the prevention of the enzymatic browning suffered by fruits and vegetables after minimal processing. The aim of this study is to determine the in vitro and in vivo activity of microwaved extracts obtained from thinned nectarines. It has been observed that in vitro the extracts obtained after the application of high microwave power levels (500, 1000 and 1500W) are mixed type inhibitors of polyphenoloxidase enzyme, showing an irreversible inactivation. This inhibition could be attributed to the Maillard reaction products formed during the microwave treatment. In vivo, a solution of 2% of the extract obtained at 1500W inhibited the enzymatic browning in minimally processed peaches for 8days of storage. PMID:26616994

  17. Control of skin colour and polyphenol oxidase activity in santol fruit by dipping in organic acid solution.

    PubMed

    Benjawan, Chutichudet; Chutichudet, P

    2009-06-01

    This laboratory experiment was carried out at the Department of Agricultural Technology, Mahasarakham University, Northeast Thailand during July and August 2008. The experiment aimed to determine an effective natural organic acid that would delay the unattractive skin browning of santol fruit, while at the same time not damaging the quality of the fruit. The experiment included a study of the fruit's polyphenol oxidase (PPO) activity, phenolic content and quinone content, as they relate to colour and a study of total soluble solid content, pH, titratable acidity and vitamin C content as they relate to fruit quality. A Completely Randomized Design (CRD) with four replications was used. Each replication consisted of 10 fruits. Santol fruit was harvested at 145 days after full bloom and dipped for 30 min in aqueous solutions of two organic acids that were used as treatments, i.e., 0% for T1 (control), 5% citric acid for T2, 5% ascorbic acid for T3, 10% citric acid for T4 and 10% ascorbic acid for T5 and stored at room temperature (28 degrees C, 90% R.H.) to investigate the effect of the acid on fruit weight, skin colour, PPO activity and other internal parameters. The results showed that the most appropriate anti-browning agent for santol fruit was found with T2. It gave the highest mean values, 57.37 and 55.95, of brightness (L*) at 4 and 10 Days After Storage (DAS), respectively. In addition, PPO activity of flesh tissue was lowest for T2 with mean values of 0.0078 to 0.0092 by 0 and 300 S, respectively. The phenolic content in the flesh tissue significantly increased with an increase in numbers ofDAS, whereas the reverse was found with the pH level in the fruits. They were lowest for T2, with mean values of 6.00, by 10 DAS. There were no significant differences among the treatments in any of the measured Total Soluble Solids (TSS), Titratable Acidity (TA) and vitamin C content. PMID:19803119

  18. Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia.

    PubMed

    Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

    2015-01-01

    In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress. PMID:26656460

  19. Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia

    PubMed Central

    Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

    2015-01-01

    In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2? were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2?, and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2? generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress. PMID:26656460

  20. Plants utilize a highly conserved system for repair of NADH and NADPH hydrates.

    PubMed

    Niehaus, Tom D; Richardson, Lynn G L; Gidda, Satinder K; ElBadawi-Sidhu, Mona; Meissen, John K; Mullen, Robert T; Fiehn, Oliver; Hanson, Andrew D

    2014-05-01

    NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast (Saccharomyces cerevisiae), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts (S)-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B6 salvage enzyme pyridoxine 5'-phosphate oxidase. Recombinant maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert (S)-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted (R)- and (S)-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5'-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea (Pisum sativum) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco (Nicotiana tabacum) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites. PMID:24599492

  1. A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level.

    PubMed

    Arciuli, Marcella; Brunetti, Adalberto; Fiocco, Daniela; Zacchino, Valentina; Centoducati, Gerardo; Aloi, Antonio; Tommasi, Raffaele; Santeramo, Arcangela; De Nitto, Emanuele; Gallone, Anna

    2015-01-01

    Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments. PMID:25449383

  2. In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.

    PubMed

    Ketchum, P A; Cambier, H Y; Frazier, W A; Madansky, C H; Nason, A

    1970-07-01

    In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit. PMID:4393266

  3. The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

    PubMed Central

    Hao, Meng-Shu; Jensen, Anna M.; Boquist, Ann-Sofie; Liu, Yun-Jun; Rasmusson, Allan G.

    2015-01-01

    NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (?100 ?M), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene. PMID:26413894

  4. Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

    PubMed

    Huang, Nana; Cheng, Xi; Hu, Wanfeng; Pan, Siyi

    2015-02-01

    The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in ?-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated