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Sample records for nadph oxidase activation

  1. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized......BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased...... inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes...

  2. Modulation of NADPH oxidase activity by known uraemic retention solutes.

    Science.gov (United States)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera; Cohen, Gerald; Schaefer, Mandy; Boehringer, Falko; Tepel, Martin; Kunkel, Desiree; Zidek, Walter; Jankowski, Joachim

    2014-08-01

    Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  3. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  4. Paraquat and maneb co-exposure induces noradrenergic locus coeruleus neurodegeneration through NADPH oxidase-mediated microglial activation

    International Nuclear Information System (INIS)

    Hou, Liyan; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Wang, Hongwei; Che, Yuning; Sun, Fuqiang; Zhou, Xueying; Zhao, Xiulan; Wang, Qingshan

    2017-01-01

    Highlights: • Microglial activation induced by paraquat and maneb precedes noradrenergic neurodegeneration in locus coeruleus. • NADPH oxidase activation contributes to microglia-mediated neuroinflammation and related noradrenergic neurodegeneration. • Inhibition of NADPH oxidase by apocynin protects noradrenergic neurons against paraquat and maneb-induced toxicity. - Abstract: Co-exposure to paraquat (PQ) and maneb (Mb) has been shown to increase the risk of Parkinson’s disease (PD) and dopaminergic (DA) neurodegeneration in the substantia nigra pars compacta (SNpc) is observed in PQ and Mb-treated experimental animals. The loss of noradrenergic locus coeruleus (LC/NE) neurons in brainstem is a common feature shared by multiple neurodegenerative diseases, including PD. However, whether PQ and Mb is able to damage LC/NE neurons remains undefined. In this study, mice treated with combined PQ and Mb displayed progressive LC/NE neurodegeneration. Time course studies revealed that the activation of microglia preceded LC/NE neurodegeneration. Mechanistically, the activation of NADPH oxidase contributed to microglial activation and subsequent LC/NE neurodegeneration. We found that PQ and Mb co-exposure induced activation of NADPH oxidase as shown by increased superoxide production and membrane translocation of p47 phox , a cytosolic subunit of NADPH oxidase. Inhibition of NADPH oxidase by apocynin, a widely used NADPH oxidase inhibitor, suppressed microglial activation and gene expressions of proinflammatory factors. Furthermore, reduced activation of nuclear factor-κB (NF-κB) pathway was observed in apocynin-treated mice. More importantly, inhibition of NADPH oxidase by apocynin afforded LC/NE neuroprotection against PQ and Mb-induced neurotoxicity. Thus, our findings revealed the critical role NADPH oxidase-mediated microglial activation in driving LC/NE neurodegeneration induced by PQ and Mb, providing new insights into the pathogenesis of environmental

  5. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

  6. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China); Wei, Ling [Beijing Center for Physical & Chemical Analysis, Beijing 100089 (China); Liu, Yutong [School of Life Science, Beijing Normal University, Beijing 100875 (China); Liao, Jieying [Department of Translational Medicine, Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences, Xiamen 361024 (China); Gao, Hui-Ming [Model Animal Research Center of Nanjing University, Nanjing 211800 (China); Zhou, Hui, E-mail: hardhui@gmail.com [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China)

    2017-05-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

  7. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    International Nuclear Information System (INIS)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin; Wei, Ling; Liu, Yutong; Liao, Jieying; Gao, Hui-Ming; Zhou, Hui

    2017-01-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm 2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm 2 ) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91 phox , p47 phox and p40 phox ); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47 phox and p67 phox translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our

  8. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

    Science.gov (United States)

    Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

    2013-09-01

    Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

  9. Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

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    Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

    2011-06-15

    Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

    2015-05-01

    Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. [The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation].

    Science.gov (United States)

    Stasia, Marie-José

    2007-05-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

  12. NADPH Oxidases: Progress and Opportunities

    OpenAIRE

    San Martin, Alejandra; Griendling, Kathy K.

    2014-01-01

    From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models...

  13. Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Quan-Guang Zhang

    Full Text Available BACKGROUND: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O(2(-, and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. METHODOLOGY/PRINCIPAL FINDINGS: The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24-96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O(2(- induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. CONCLUSIONS/SIGNIFICANCE: As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

  14. Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea.

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    Deepti Nair

    Full Text Available In rodents, exposure to intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox(_/Y and wild-type littermates. On a standard place training task, gp91phox(_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA controls, while no changes emerged in gp91phox(_/Y mice. Additionally, wild-type mice, but not gp91phox(_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.

  15. Blockade of TGF-β 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats

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    José Luis Miguel-Carrasco

    2012-01-01

    Full Text Available NADPH oxidases constitute a major source of superoxide anion (⋅O2 - in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

  16. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance

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    Giang T. Nguyen

    2017-08-01

    Full Text Available Reactive oxygen species (ROS generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs, toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs. Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation

  17. Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane.

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    El-Benna, Jamel; Dang, Pham My-Chan; Gougerot-Pocidalo, Marie-Anne

    2008-07-01

    Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of

  18. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

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    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

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

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    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 0.1μM fMLF and 1μM 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

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    Musicki, Biljana; Bivalacqua, Trinity J; Champion, Hunter C; Burnett, Arthur L

    2014-02-01

    Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P penis. Sildenafil treatment of WT mice did not affect any of these parameters. Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. © 2013 International Society for Sexual Medicine.

  1. Functional heterogeneity of NADPH oxidase-mediated contractions to endothelin with vascular aging.

    Science.gov (United States)

    Meyer, Matthias R; Barton, Matthias; Prossnitz, Eric R

    2014-11-24

    Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1. Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L). In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (prenal artery and aorta, respectively (pAging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, page-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    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 (O 3 ) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O 3 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 O 3 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 O 3 , determined from the mRNA levels of the major allergens. We conclude that O 3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: → O 3 reduces the viability of ragweed pollen. → ROS and allergens of ragweed pollen were not affected by O 3 exposure. → O 3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. → O 3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, Stefania, E-mail: spas@unipg.it [Department of Applied Biology, University of Perugia, Perugia (Italy); Tedeschini, Emma; Frenguelli, Giuseppe [Department of Applied Biology, University of Perugia, Perugia (Italy); Wopfner, Nicole; Ferreira, Fatima [Department of Molecular Biology, CD Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg (Austria); D' Amato, Gennaro [Division of Respiratory and Allergic Diseases, ' A. Cardarelli' High Speciality Hospital, Naples (Italy); Ederli, Luisa [Department of Applied Biology, University of Perugia, Perugia (Italy)

    2011-10-15

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} 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 O{sub 3} 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 O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  4. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

  5. Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

    Science.gov (United States)

    Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

    2017-04-01

    We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression.

    Science.gov (United States)

    Calabriso, Nadia; Massaro, Marika; Scoditti, Egeria; D'Amore, Simona; Gnoni, Antonio; Pellegrino, Mariangela; Storelli, Carlo; De Caterina, Raffaele; Palasciano, Giuseppe; Carluccio, Maria Annunziata

    2016-02-01

    Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1-10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (Pextra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 2 ·- 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 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 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 phox , p67 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 phox siRNA. Exposure of MPMVEC obtained from gp91 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

  8. NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

    Science.gov (United States)

    Vlahos, Ross; Selemidis, Stavros

    2014-12-01

    Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

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

    Science.gov (United States)

    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.

  10. Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes

    Directory of Open Access Journals (Sweden)

    Gi Soo Youn

    2017-08-01

    Full Text Available Histone deacetylase 6 (HDAC6 likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat activates NADPH oxidase resulting in generation of reactive oxygen species (ROS, leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation. Keywords: HIV-1 Tat, HDAC6, NADPH oxidase, ROS, Inflammation, Astrocytes

  11. NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L. seedlings exposed to cadmium

    Directory of Open Access Journals (Sweden)

    Jagna Chmielowska-Bąk

    2017-06-01

    Full Text Available Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS. Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.

  12. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    Science.gov (United States)

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Landreth Gary E

    2006-11-01

    Full Text Available Abstract Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

  14. Two X-linked chronic granulomatous disease patients with unusual NADPH oxidase properties

    NARCIS (Netherlands)

    Wolach, Baruch; Broides, Arnon; Zeeli, Tal; Gavrieli, Ronit; de Boer, Martin; van Leeuwen, Karin; Levy, Jacov; Roos, Dirk

    2011-01-01

    Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two

  15. Peroxisomal Polyamine Oxidase and NADPH-Oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Efthimios A. Andronis

    2014-04-01

    Full Text Available Homeostasis of reactive oxygen species (ROS in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd and spermine (Spm to putrescine (Put and Spd, respectively is catalyzed by two peroxisomal PA oxidases (AtPAO. However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI. Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2.-, but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX. On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2.-. These results suggest that the ratio of O2.-/H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2.- by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.

  16. H2O2 and NADPH oxidases involve in regulation of 2-(2-phenylethyl)chromones accumulation during salt stress in Aquilaria sinensis calli.

    Science.gov (United States)

    Wang, Xiaohui; Dong, Xianjuan; Feng, Yingying; Liu, Xiao; Wang, Jinling; Zhang, Zhongxiu; Li, Jun; Zhao, Yunfang; Shi, Shepo; Tu, Pengfei

    2018-04-01

    2-(2-Phenylethyl)chromones are the main compounds responsible for the quality of agarwood, which is widely used in traditional medicines, incenses and perfumes. H 2 O 2 and NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs) mediate diverse physiological and biochemical processes in environmental stress responses. However, little is known about the function of H 2 O 2 and NADPH oxidases in 2-(2-phenylethyl)chromones accumulation. In this study, we found that salt stress induced a transient increase in content of H 2 O 2 and 2-(2-phenylethyl)chromones accumulation in Aquilaria sinensis calli. Exogenous H 2 O 2 remarkably decreased the production of 2-(2-phenylethyl)chromones, while dimethylthiourea (DMTU), a scavenger of H 2 O 2 , significantly increased 2-(2-phenylethyl)chromones accumulation in salt treated calli. Three new H 2 O 2 -generating genes, named AsRbohA-C, were isolated and characterized from A. sinensis. Salt stress also induced a transient increase in AsRbohA-C expression and NADPH oxidase activity. Furthermore, exogenous H 2 O 2 increased AsRbohA-C expression and NADPH oxidase activity, while DMTU inhibited AsRbohA-C expression and NADPH oxidase activity under salt stress. Moreover, diphenylene iodonium (DPI), the inhibitor of NADPH oxidases, reduced AsRbohA-C expression and NADPH oxidase activity, but significantly induced 2-(2-phenylethyl)chromones accumulation during salt stress. These results clearly demonstrated the central role of H 2 O 2 and NADPH oxidases in regulation of salt-induced 2-(2-phenylethyl)chromones accumulation in A. sinensis calli. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. NADPH Oxidase Activity in Cerebral Arterioles Is a Key Mediator of Cerebral Small Vessel Disease-Implications for Prevention.

    Science.gov (United States)

    McCarty, Mark F

    2015-04-15

    Cerebral small vessel disease (SVD), a common feature of brain aging, is characterized by lacunar infarcts, microbleeds, leukoaraiosis, and a leaky blood-brain barrier. Functionally, it is associated with cognitive decline, dementia, depression, gait abnormalities, and increased risk for stroke. Cerebral arterioles in this syndrome tend to hypertrophy and lose their capacity for adaptive vasodilation. Rodent studies strongly suggest that activation of Nox2-dependent NADPH oxidase activity is a crucial driver of these structural and functional derangements of cerebral arterioles, in part owing to impairment of endothelial nitric oxide synthase (eNOS) activity. This oxidative stress may also contribute to the breakdown of the blood-brain barrier seen in SVD. Hypertension, aging, metabolic syndrome, smoking, hyperglycemia, and elevated homocysteine may promote activation of NADPH oxidase in cerebral arterioles. Inhibition of NADPH oxidase with phycocyanobilin from spirulina, as well as high-dose statin therapy, may have potential for prevention and control of SVD, and high-potassium diets merit study in this regard. Measures which support effective eNOS activity in other ways-exercise training, supplemental citrulline, certain dietary flavonoids (as in cocoa and green tea), and capsaicin, may also improve the function of cerebral arterioles. Asian epidemiology suggests that increased protein intakes may decrease risk for SVD; conceivably, arginine and/or cysteine-which boosts tissue glutathione synthesis, and can be administered as N-acetylcysteine-mediate this benefit. Ameliorating the risk factors for SVD-including hypertension, metabolic syndrome, hyperglycemia, smoking, and elevated homocysteine-also may help to prevent and control this syndrome, although few clinical trials have addressed this issue to date.

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: shakim@lgls.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)

    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 μM. 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.

  19. NADPH Oxidase Activity in Cerebral Arterioles Is a Key Mediator of Cerebral Small Vessel Disease—Implications for Prevention

    Directory of Open Access Journals (Sweden)

    Mark F. McCarty

    2015-04-01

    Full Text Available Cerebral small vessel disease (SVD, a common feature of brain aging, is characterized by lacunar infarcts, microbleeds, leukoaraiosis, and a leaky blood-brain barrier. Functionally, it is associated with cognitive decline, dementia, depression, gait abnormalities, and increased risk for stroke. Cerebral arterioles in this syndrome tend to hypertrophy and lose their capacity for adaptive vasodilation. Rodent studies strongly suggest that activation of Nox2-dependent NADPH oxidase activity is a crucial driver of these structural and functional derangements of cerebral arterioles, in part owing to impairment of endothelial nitric oxide synthase (eNOS activity. This oxidative stress may also contribute to the breakdown of the blood-brain barrier seen in SVD. Hypertension, aging, metabolic syndrome, smoking, hyperglycemia, and elevated homocysteine may promote activation of NADPH oxidase in cerebral arterioles. Inhibition of NADPH oxidase with phycocyanobilin from spirulina, as well as high-dose statin therapy, may have potential for prevention and control of SVD, and high-potassium diets merit study in this regard. Measures which support effective eNOS activity in other ways—exercise training, supplemental citrulline, certain dietary flavonoids (as in cocoa and green tea, and capsaicin, may also improve the function of cerebral arterioles. Asian epidemiology suggests that increased protein intakes may decrease risk for SVD; conceivably, arginine and/or cysteine—which boosts tissue glutathione synthesis, and can be administered as N-acetylcysteine—mediate this benefit. Ameliorating the risk factors for SVD—including hypertension, metabolic syndrome, hyperglycemia, smoking, and elevated homocysteine—also may help to prevent and control this syndrome, although few clinical trials have addressed this issue to date.

  20. Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries.

    Science.gov (United States)

    Simplicio, Janaina A; Hipólito, Ulisses Vilela; Vale, Gabriel Tavares do; Callera, Glaucia Elena; Pereira, Camila André; Touyz, Rhian M; Tostes, Rita de Cássia; Tirapelli, Carlos R

    2016-11-01

    The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism. O mecanismo da disfunção vascular induzido pelo consumo de etanol não é totalmente compreendido. Justifica-se, assim a identificação de mecanismos bioquímicos e moleculares que poderiam explicar tais efeitos. Investigar se a ingestão aguda de etanol ativa a via vascular RhoA/Rho quinase

  1. Desbalanço redox: NADPH oxidase como um alvo terapêutico no manejo cardiovascular Desbalance redox: NADPH oxidasa como un objetivo terapéutico en el manejo cardiovascular Redox unbalance: NADPH oxidase as therapeutic target in blood pressure control

    Directory of Open Access Journals (Sweden)

    Luiza A. Rabêlo

    2010-05-01

    la mayor fuente de esas especies reactivas en la vasculatura. En condiciones fisiológicas, la formación y eliminación de esas sustancias aparecen balanceadas en la pared vascular. Durante el desbalance redox, sin embargo, hay un aumento en la actividad de la NADPH oxidasa y predominio de agentes prooxidantes, superando la capacidad de defensa orgánica antioxidante. Además de ello, tal hiperactividad enzimática reduce la biodisponibilidad del óxido nítrico, crucial para la vasodilatación y el mantenimiento de la función vascular normal. A pesar de que la NADPH oxidasa se relaciona directamente con la disfunción endotelial, fue primeramente descrita por su expresión en fagocitos, en los que su actividad enzimática determina la eficacia de los mecanismos de defensa orgánica contra patógenos. Las diferencias sutiles entre las unidades estructurales de las NADPH oxidasas, dependiendo del tipo celular que las expresa, pueden tener implicaciones terapéuticas, permitiendo la inhibición selectiva del desequilibrio redox inducido por la NADPH oxidasa, sin comprometer, con todo, su participación en las vías fisiológicas de señalización celular que garantizan la protección contra microorganismos.Several studies refer to reactive oxygen and nitrogen species (RONS as important agents in the pathogenesis of a number of heart diseases, including high blood pressure, arteriosclerosis and heart failure. Such species are highly bioactive molecules and a short life due chiefly to reduction of molecular oxygen. The enzyme complex of NADPH oxidase is the main source of these reactive species in vascular system. Under physiological conditions, formation and elimination of these substances seem balanced in vascular wall. During redox Unbalance, nonetheless, there is increase in NADPH oxidase activity and predominance of pro-oxidizing agents, surpassing the anti-oxidant capacity of the organism self-defense. Besides this, such enzyme hyperactivity reduces the

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  3. Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

    Science.gov (United States)

    Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

    2015-08-01

    Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

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    Gábor Sirokmány

    2018-06-01

    Full Text Available Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels. Keywords: Peroxidasin, NADPH oxidase, Hydrogen peroxide, Collagen IV, Sulfilimine

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

    Science.gov (United States)

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

    2011-10-01

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) 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 O(3) 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 O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

    Science.gov (United States)

    Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

    2009-09-01

    Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

  7. Kaempferol modulates pro-inflammatory NF-κB activation by suppressing advanced glycation endproducts-induced NADPH oxidase

    Science.gov (United States)

    Kim, Ji Min; Lee, Eun Kyeong; Kim, Dae Hyun; Yu, Byung Pal

    2010-01-01

    Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-ĸB). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-ĸB and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-κB activity and NF-ĸB-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-κB activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-κB signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies. PMID:20431987

  8. Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Peiying Yu

    2014-01-01

    Full Text Available NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5 gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT and hypertensive subjects (HT. We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group. Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA, effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8 in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8% than HT (−14.8±1.8. In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

  9. Pharmacological inhibition of NADPH oxidase protects against cisplatin induced nephrotoxicity in mice by two step mechanism.

    Science.gov (United States)

    Wang, Yimin; Luo, Xiao; Pan, Hao; Huang, Wei; Wang, Xueping; Wen, Huali; Shen, Kezhen; Jin, Baiye

    2015-09-01

    Cisplatin induced nephrotoxicity is primarily caused by ROS (Reactive Oxygen Species) induced proximal tubular cell death. NADPH oxidase is major source of ROS production by cisplatin. Here, we reported that pharmacological inhibition of NADPH oxidase by acetovanillone (obtained from medicinal herb Picrorhiza kurroa) led to reduced cisplatin nephrotoxicity in mice. In this study we used various molecular biology and biochemistry methods a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin. Cisplatin-induced nephrotoxicity was evident by histological damage from loss of the tubular structure. The damage was also marked by the increase in blood urea nitrogen, creatinine, protein nitration as well as cell death markers such as caspase 3/7 activity and DNA fragmentation. Tubular cell death by cisplatin led to pro-inflammatory response by production of TNFα and IL1β followed by leukocyte/neutrophil infiltration which resulted in new wave of ROS involving more NADPH oxidases. Cisplatin-induced markers of kidney damage such as oxidative stress, cell death, inflammatory cytokine production and nephrotoxicity were attenuated by acetovanillone. In addition to that, acetovanillone enhanced cancer cell killing efficacy of cisplatin. Thus, pharmacological inhibition of NADPH oxidase can be protective for cisplatin-induced nephrotoxicity in mice. Copyright © 2015. Published by Elsevier Ltd.

  10. NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

    International Nuclear Information System (INIS)

    Newaz, Mohammad; Blanton, Ahmad; Fidelis, Paul; Oyekan, Adebayo

    2005-01-01

    Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). L-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p 2+ -dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53-7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and

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

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available 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-κB 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-κB 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-κB 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-κB pathways in neutrophils.

  12. Amyloid β induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase- and mitochondria-dependent ROS production.

    Science.gov (United States)

    Wang, Ke; Yao, Yong; Zhu, Xue; Zhang, Kai; Zhou, Fanfan; Zhu, Ling

    2017-06-01

    Amyloid β (Aβ)-induced chronic inflammation is believed to be a key pathogenic process in early-stage age-related macular degeneration (AMD). Nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation triggered by Aβ is responsible for retinal pigment epithelium (RPE) dysfunction in the onset of AMD; however, the detailed molecular mechanism remains unclear. In this study, we investigated the involvement of NADPH oxidase- and mitochondria-derived reactive oxygen species (ROS) in the process of Aβ 1-40 -induced NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. The results showed that Aβ 1-40 could induce excessive ROS generation, MAPK/NF-κB signaling activation and subsequently NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. Furthermore, the inductive effect of Aβ 1-40 on NLRP3 inflammasome activation was mediated in a manner dependent on NADPH oxidase- and mitochondria-derived ROS. Our findings may provide a novel insight into the molecular mechanism by which Aβ contributes to the early-stage AMD. © 2016 Wiley Periodicals, Inc.

  13. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis

    Science.gov (United States)

    Kwak, June M.; Mori, Izumi C.; Pei, Zhen-Ming; Leonhardt, Nathalie; Torres, Miguel Angel; Dangl, Jeffery L.; Bloom, Rachel E.; Bodde, Sara; Jones, Jonathan D.G.; Schroeder, Julian I.

    2003-01-01

    Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca2+ increases and ABA- activation of plasma membrane Ca2+-permeable channels in guard cells. Exogenous H2O2 rescues both Ca2+ channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction. PMID:12773379

  14. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    Science.gov (United States)

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  15. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

    Directory of Open Access Journals (Sweden)

    Elaine Hatanaka

    Full Text Available The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.

  16. Interferon gamma/NADPH oxidase defence system in immunity and cancer

    Czech Academy of Sciences Publication Activity Database

    Hodný, Zdeněk; Reiniš, Milan; Hubáčková, Soňa; Vašicová, Pavla; Bartek, Jiří

    -, 01 Sep (2015) ISSN 2162-4011 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : IFNγ * NADPH oxidase * immunity * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.266, year: 2014

  17. Role of catechins on ET-1 induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: Determination of the probable mechanism by molecular docking studies.

    Science.gov (United States)

    Chakraborti, Sajal; Sarkar, Jaganmay; Bhuyan, Rajabrata; Chakraborti, Tapati

    2017-12-05

    Treatment of human pulmonary artery smooth muscle cells with ET-1 stimulated PLD and NADPH oxidase activities, which were inhibited upon pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor) and EGCG & ECG (catechins having galloyl group), but not EGC & EC (catechins devoid of galloyl group). Herein, we determined the probable mechanism by which the galloyl group containing catechins inhibit ET-1 induced stimulation of PLD activity by molecular docking analyses based on our biochemical studies. ET-1 induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf-6 and cytohesin-1 were associated in the cell membrane, which was not inhibited by the catechins during ET-1 treatment to the cells. However, EGCG and ECG inhibited binding of GTPγS with Arf-6 even in presence of cytohesin-1. The molecular docking analyses revealed that the galloyl group containing catechins (EGCG/ECG) with cytohesin1-Arf6GDP, but not the non-galloyl-containing catechins (EGC and EC), prevents GDP/GTP exchange in Arf-6 which seems to be an important mechanism for inhibition of ET-1 induced activation of PLD and subsequently increase in NADPH oxidase activities.

  18. Deciphering the role of NADPH oxidase in complex interactions between maize (Zea mays L.) genotypes and cereal aphids.

    Science.gov (United States)

    Sytykiewicz, Hubert

    2016-07-22

    Plant NADPH oxidases (NOXs) encompass a group of membrane-bound enzymes participating in formation of reactive oxygen species (ROS) under physiological conditions as well as in response to environmental stressors. The purpose of the survey was to unveil the role of NADPH oxidase in pro-oxidative responses of maize (Zea mays L.) seedling leaves exposed to cereal aphids' infestation. The impact of apteral females of bird cherry-oat aphid (Rhopalosiphum padi L.) and grain aphid (Sitobion avenae F.) feeding on expression levels of all four NADPH oxidase genes (rbohA, rbohB, rbohC, rbohD) and total activity of NOX enzyme in maize plants were investigated. In addition, inhibitory effect of diphenylene iodonium (DPI) pre-treatment on NOX activity and hydrogen peroxide content in aphid-stressed maize seedlings was studied. Leaf infestation biotests were accomplished on 14-day-old seedlings representing two aphid-resistant varieties (Ambrozja and Waza) and two aphid-susceptible ones (Tasty Sweet and Złota Karłowa). Insects' attack led to profound upregulation of rbohA and rbohD genes in tested host plants, lower elevations were noted in level of rbohB mRNA, whereas abundance of rbohC transcript was not significantly altered. It was uncovered aphid-induced enhancement of NOX activity in examined plants. Higher increases in expression of all investigated rboh genes and activity of NADPH oxidase occurred in tissues of more resistant maize cultivars than in susceptible ones. Furthermore, DPI treatment resulted in strong reduction of NOX activity and H2O2 accumulation in aphid-infested Z. mays plants, thus evidencing circumstantial role of the enzyme in insect-elicited ROS generation. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

    Science.gov (United States)

    Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

    2015-10-01

    To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

  20. Safrole oxide induces neuronal apoptosis through inhibition of integrin beta4/SOD activity and elevation of ROS/NADPH oxidase activity.

    Science.gov (United States)

    Su, Le; Zhao, BaoXiang; Lv, Xin; Wang, Nan; Zhao, Jing; Zhang, ShangLi; Miao, JunYing

    2007-02-20

    Neuronal apoptosis is a very important event in the development of the central nervous system (CNS), but the underlying mechanisms remain to be elucidated. We have previously shown that safrole oxide, a small molecule, induces integrin beta4 expression and promotes apoptosis in vascular endothelial cells. In this study, the effects of safrole oxide on cell growth and apoptosis have been examined in primary cultures of mouse neurons. Safrole oxide was found to significantly inhibit neuronal cell growth and to induce apoptosis. The inhibitory and apoptotic activities of safrole oxide followed a dose- and time-dependent manner. Interestingly, the expression of integrin beta4 was significantly inhibited with safrole oxide treatment. Furthermore, safrole oxide dramatically increases the level of intracellular reactive oxygen species (ROS) and the activity of NADPH oxidase. Moreover, manganese-dependent superoxide dismutase (MnSOD) activity was decreased significantly with safrole oxide treatment. Our study thus demonstrates that safrole oxide induces neuronal apoptosis through integrin beta4, ROS, NADPH, and MnSOD.

  1. Effect of a heme oxygenase-1 inducer on NADPH oxidase ...

    African Journals Online (AJOL)

    Effect of a heme oxygenase-1 inducer on NADPH oxidase expression in ... and immunohistochemistry of hepatic NOX1 and NOX4 were investigated in week 4. ... (HO-1 inhibitor) administration caused upregulation of NOX gene expression ...

  2. Resveratrol prevents high glucose-induced epithelial-mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway.

    Science.gov (United States)

    He, Ting; Guan, Xu; Wang, Song; Xiao, Tangli; Yang, Ke; Xu, Xinli; Wang, Junping; Zhao, Jinghong

    2015-02-15

    Resveratrol (RSV) is reported to have renoprotective activity against diabetic nephropathy, while the mechanisms underlying its function have not been fully elucidated. In this study, we investigate the effect and related mechanism of RSV against high glucose-induced epithelial to mesenchymal transition (EMT) in human tubular epithelial cells (HK-2). A typical EMT is induced by high glucose in HK-2 cells, accompanied by increased levels of reactive oxygen species (ROS). RSV exhibits a strong ability to inhibit high glucose-induced EMT by decreasing intracellular ROS levels via down-regulation of NADPH oxidase subunits NOX1 and NOX4. The activation of extracellular signal-regulated kinase (ERK1/2) is found to be involved in high glucose-induced EMT in HK-2 cells. RSV, like NADPH oxidase inhibitor diphenyleneiodonium, can block ERK1/2 activation induced by high glucose. Our results demonstrate that RSV is a potent agent against high glucose-induced EMT in renal tubular cells via inhibition of NADPH oxidase/ROS/ERK1/2 pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. NADPH oxidase and redox status in amygdala, hippocampus and cortex of male Wistar rats in an animal model of post-traumatic stress disorder.

    Science.gov (United States)

    Petrovic, Romana; Puskas, Laslo; Jevtic Dozudic, Gordana; Stojkovic, Tihomir; Velimirovic, Milica; Nikolic, Tatjana; Zivkovic, Milica; Djorovic, Djordje J; Nenadovic, Milutin; Petronijevic, Natasa

    2018-05-26

    Post-traumatic stress disorder (PTSD) is a highly prevalent and impairing disorder. Oxidative stress is implicated in its pathogenesis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of free radicals. The aim of the study was to assess oxidative stress parameters, activities of respiratory chain enzymes, and the expression of NADPH oxidase subunits (gp91phox, p22phox, and p67phox) in the single prolonged stress (SPS) animal model of PTSD. Twenty-four (12 controls; 12 subjected to SPS), 9-week-old, male Wistar rats were used. SPS included physical restraint, forced swimming, and ether exposure. The rats were euthanized seven days later. Cortex, hippocampus, amygdala, and thalamus were dissected. Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), Complex I, and cytochrome C oxidase were measured using spectrophotometric methods, while the expression of NADPH oxidase subunits was determined by Western blot. Increased MDA and decreased GSH concentrations were found in the amygdala and hippocampus of the SPS rats. SOD activity was decreased in amygdala and GPx was decreased in hippocampus. Increased expression of the NADPH oxidase subunits was seen in amygdala, while mitochondrial respiratory chain enzyme expression was unchanged both in amygdala and hippocampus. In the cortex concentrations of MDA and GSH were unchanged despite increased Complex I and decreased GPx, while in the thalamus no change of any parameter was noticed. We conclude that oxidative stress is present in hippocampus and amygdala seven days after the SPS procedure. NADPH oxidase seems to be a main source of free radicals in the amygdala.

  4. NADPH oxidase: an enzyme for multicellularity?

    Science.gov (United States)

    Lalucque, Hervé; Silar, Philippe

    2003-01-01

    Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.

  5. Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

    Directory of Open Access Journals (Sweden)

    Lee I-Ta

    2012-11-01

    Full Text Available Abstract Background In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4 activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1, which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs. Results We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88, Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2, and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone, NADPH oxidase [apocynin (APO or diphenyleneiodonium chloride (DPI], c-Src (PP1, p38 MAPK (SB202190, and p300 (GR343. LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody. Conclusions In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may

  6. Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase.

    Science.gov (United States)

    Nie, Huibin; Xue, Xia; Liu, Gang; Guan, Guangju; Liu, Haiying; Sun, Lina; Zhao, Long; Wang, Xueling; Chen, Zhixin

    2016-01-01

    Nitroalkene derivative of oleic acid (OA-NO 2 ), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO 2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO 2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO 2 (1.25 μM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO 2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO 2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22 phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO 2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

  7. The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

    Science.gov (United States)

    Jakubowska, Dagmara; Janicka, Małgorzata

    2017-11-01

    The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H + -ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H + -ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H + -ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H + -ATPase and NADPH oxidase are key factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. NADPH oxidase is not an essential mediator of oxidative stress or liver injury in murine MCD diet-induced steatohepatitis.

    Science.gov (United States)

    dela Peña, Aileen; Leclercq, Isabelle A; Williams, Jacqueline; Farrell, Geoffrey C

    2007-02-01

    Hepatic oxidative stress is a key feature of metabolic forms of steatohepatitis, but the sources of pro-oxidants are unclear. The NADPH oxidase complex is critical for ROS generation in inflammatory cells; loss of any one component (e.g., gp91phox) renders NADPH oxidase inactive. We tested whether activated inflammatory cells contribute to oxidant stress in steatohepatitis. gp91phox-/- and wildtype (wt) mice were fed a methionine and choline-deficient (MCD) diet. Serum ALT, hepatic triglycerides, histopathology, lipid peroxidation, activation of NF-kappaB, expression of NF-kappaB-regulated genes and macrophage chemokines were measured. After 10 days of MCD dietary feeding, gp91phox-/- and wt mice displayed equivalent hepatocellular injury. After 8 weeks, there were fewer activated macrophages in livers of gp91phox-/- mice than controls, despite similar mRNA levels for MCP and MIP chemokines, but fibrosis was similar. NF-kappaB activation and increased expression of ICAM-1, TNF-alpha and COX-2 mRNA were evident in both genotypes, but in gp91phox-/- mice, expression of these genes was confined to hepatocytes. A functional NADPH oxidase complex does not contribute importantly to oxidative stress in this model and therefore is not obligatory for induction or perpetuation of dietary steatohepatitis.

  9. Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Pedersen, G

    2003-01-01

    Superoxide (O(2)(-)) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells....

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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 phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47 phox was translocated to the cell membrane and localized with p22 phox and gp91 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.

  11. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    Science.gov (United States)

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  12. Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

    Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

  13. Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain?

    OpenAIRE

    Nisimoto, Yukio; Jackson, Heather M.; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J. David

    2010-01-01

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47 phox and p67 phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K m for NADPH of 55 ? 10 ?M. The concentration of Nox4 in cell lysates was esti...

  14. NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases

    Czech Academy of Sciences Publication Activity Database

    Potocký, Martin; Pejchar, Přemysl; Gutkowska, Malgorzata; Jiménez-Quesada, M. J.; Potocká, Andrea; Alché, J.; Kost, B.; Žárský, Viktor

    2012-01-01

    Roč. 169, č. 16 (2012), s. 1654-1663 ISSN 0176-1617 R&D Projects: GA ČR GP522/09/P299 Institutional research plan: CEZ:AV0Z50380511 Keywords : Pollen tube * Tip growth * NADPH oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.699, year: 2012

  15. Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis

    DEFF Research Database (Denmark)

    Roepstorff, Kirstine; Rasmussen, Izabela Zorawska; Sawada, Makoto

    2008-01-01

    dominant-positive mutants enhanced, whereas dominant-negative mutants inhibited, NADPH oxidase-mediated superoxide generation following formyl-methionyl-leucylphenylalanine or phorbol 12-myristate 13-acetate stimulation. Both Rac1 and the GTP exchange factor VAV1 were required as upstream signaling......The p21-activated kinase-1 (PAK1) is best known for its role in the regulation of cytoskeletal and transcriptional signaling pathways. We show here in the microglia cell line Ra2 that PAK1 regulates NADPH oxidase (NOX-2) activity in a stimulus-specific manner. Thus, conditional expression of PAK1...... proteins in the formyl-methionyl-leucyl-phenylalanine-induced activation of endogenous PAK1. In contrast, PAK1 mutants had no effect on superoxide generation downstream of FcgammaR signaling during phagocytosis of IgG-immune complexes. We further present evidence that the effect of PAK1 on the respiratory...

  16. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

    Science.gov (United States)

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2014-07-10

    Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

  17. Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase, extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun N-terminal kinase downregulation in obese rats.

    Science.gov (United States)

    Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal

    2015-02-01

    Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.

  18. Novel p47phox-related organizers regulate NADPH oxidase 1 (Nox1) activity and localization

    Science.gov (United States)

    Gianni, Davide; Diaz, Begoña; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A.; Bokoch, Gary M.

    2010-01-01

    The mechanisms that determine localized formation of reactive oxygen species (ROS) via NADPH oxidases (Nox) in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 and Tks5 are functional members of a p47phox-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (vs. Nox2 and Nox4) activity in reconstituted cellular systems, and interact with the NoxA1 activator protein through an SH3-mediated interaction. Endogenous Tks4 is required for Rac GTPase-dependent ROS production by DLD1 colon cancer cells. Tks4 recruits Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent novel members of an organizer superfamily that link Nox to localized ROS formation. PMID:19755710

  19. RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-κB pathway in human endothelial cells.

    Science.gov (United States)

    Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

    2013-12-13

    An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

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

    Science.gov (United States)

    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 IκBα 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‑κB (NF‑κB) pathway, providing a new perspective for the clinical treatment of DN.

  1. Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity.

    Science.gov (United States)

    Gianni, Davide; Diaz, Begoña; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A; Bokoch, Gary M

    2009-09-15

    The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47(phox)-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain-mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase- and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation.

  2. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

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    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...... dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton....

  3. Long-Term Effects of Maternal Deprivation on Redox Regulation in Rat Brain: Involvement of NADPH Oxidase

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    Branka Marković

    2017-01-01

    Full Text Available Maternal deprivation (MD causes perinatal stress, with subsequent behavioral changes which resemble the symptoms of schizophrenia. The NADPH oxidase is one of the major generators of reactive oxygen species, known to play a role in stress response in different tissues. The aim of this study was to elucidate the long-term effects of MD on the expression of NADPH oxidase subunits (gp91phox, p22phox, p67phox, p47phox, and p40phox. Activities of cytochrome C oxidase and respiratory chain Complex I, as well as the oxidative stress parameters using appropriate spectrophotometric techniques were analyzed. Nine-day-old Wistar rats were exposed to a 24 h maternal deprivation and sacrificed at young adult age. The structures affected by perinatal stress, cortex, hippocampus, thalamus, and caudate nuclei were investigated. The most prominent findings were increased expressions of gp91phox in the cortex and hippocampus, increased expression of p22phox and p40phox, and decreased expression of gp91phox, p22phox, and p47phox in the caudate nuclei. Complex I activity was increased in all structures except cortex. Content of reduced glutathione was decreased in all sections while region-specific changes of other oxidative stress parameters were found. Our results indicate the presence of long-term redox alterations in MD rats.

  4. NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

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    Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C; Dominik, Malte J; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas; Steinmetz, Ivo

    2017-02-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91 phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91 phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91 phox KO mice. Only infected gp91 phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis. Copyright © 2017 American Society for Microbiology.

  5. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

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    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

    2016-01-01

    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  6. New insights into the roles of NADPH oxidases in sexual development and ascospore germination in Sordaria macrospora.

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    Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

    2014-03-01

    NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of nox2, lacking the NADPH oxidase 2 gene, nor1, and transcription factor deletion mutant ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi.

  7. The effect of orexin-A on cardiac dysfunction mediated by NADPH oxidase-derived superoxide anion in ventrolateral medulla.

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    Jun Chen

    Full Text Available Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA and projecting to the brain sites of rostral ventrolateral medulla (RVLM, involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI by releasing NAD(PH oxidase-derived superoxide anion (O2 (- generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR, heart rate variability (HRV, mean arterial pressure (MAP and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX1R increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX1R positive cells and NAD(PH oxidative subunit gp91phox or p47phox-immunoreactive (IR cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O2 (- production and mRNA expression of NAD(PH oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX1R and OX2R (SB-408124 and TCS OX2 29, respectively or apocynin (APO, an inhibitor of NAD(PH oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(PH-derived O

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

    DEFF Research Database (Denmark)

    Peleli, Maria; Al-Mashhadi, Ammar; Yang, Ting

    2016-01-01

    Hydronephrosis is associated with development of salt-sensitive hypertension. Studies suggest that increased sympathetic nerve activity (SNA) and oxidative stress play important roles in renovascular hypertension. This study aimed to investigate the link between renal SNA and NADPH oxidase (NOX......) regulation in the development of hypertension in rats with hydronephrosis. Hydronephrosis was induced by partial unilateral ureteral obstruction (PUUO) in young rats. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high and low salt diets. Renal...

  9. A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

    DEFF Research Database (Denmark)

    Dang, Pham My-Chan; Stensballe, Allan; Boussetta, Tarek

    2006-01-01

    mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM......Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem...

  10. Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

    Science.gov (United States)

    Blanca, Antonio J; Ruiz-Armenta, María V; Zambrano, Sonia; Salsoso, Rocío; Miguel-Carrasco, José L; Fortuño, Ana; Revilla, Elisa; Mate, Alfonso; Vázquez, Carmen M

    2016-10-01

    Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-α/NADPH Oxidase Signaling Pathway

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    Lishan Zhou

    2013-01-01

    Full Text Available Hu-Lu-Ba-Wan (HLBW is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH and protein kinase C-alpha (PKC-α related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-α in renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-α/NADPH oxidase signaling pathway.

  12. NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs.

    Science.gov (United States)

    Saez, Fara; Hong, Nancy J; Garvin, Jeffrey L

    2018-05-01

    Luminal flow augments Na + reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na + , playing a key role in its homeostasis. Whether flow elevations enhance Na + -K + -2Cl - cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

  13. ATL9, a RING zinc finger protein with E3 ubiquitin ligase activity implicated in chitin- and NADPH oxidase-mediated defense responses.

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    Marta Berrocal-Lobo

    2010-12-01

    Full Text Available 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.

  14. Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells.

    Science.gov (United States)

    Oliveira, Jessica Silva Santos de; Santos, Gabriela da Silva; Moraes, João Alfredo; Saliba, Alessandra Mattos; Barja-Fidalgo, Thereza Christina; Mattos-Guaraldi, Ana Luíza; Nagao, Prescilla Emy

    2018-01-01

    BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

  15. Oral treatment with the NADPH oxidase antagonist apocynin mitigates clinical and pathological features of parkinsonism in the MPTP marmoset model

    DEFF Research Database (Denmark)

    Philippens, Ingrid H C H M; Wubben, Jacqueline A; Finsen, Bente

    2013-01-01

    models, the conditions for metabolic activation of apocynin and inhibition of microglia NADPH oxidase are in place. Marmoset monkeys received oral apocynin (100 mg/kg; p.o.) (n = 5) or Gum Arabica (controls; n = 5) three times daily until the end of the study, starting 1 week before PD induction...

  16. Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions.

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    Keith D Rochfort

    Full Text Available Blood-brain barrier (BBB dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.The present study employs human brain microvascular endothelial cells (HBMvECs to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5 to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.In response to treatment with either TNF-α or IL-6 (0-100 ng/ml, 0-24 hrs, our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766.A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the

  17. Urotensin II inhibits skeletal muscle glucose transport signaling pathways via the NADPH oxidase pathway.

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    Hong-Xia Wang

    Full Text Available Our previous studies have demonstrated that the urotensin (UII and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM, but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.

  18. ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

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    Shin-Ei Cheng

    Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

  19. NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

    Science.gov (United States)

    Song, Hongman; Vijayasarathy, Camasamudram; Zeng, Yong; Marangoni, Dario; Bush, Ronald A.; Wu, Zhijian; Sieving, Paul A.

    2016-01-01

    Purpose The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1. Methods Transgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. Results Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas. Conclusions Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway. PMID:27233035

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

    International Nuclear Information System (INIS)

    Riganti, Chiara; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario

    2008-01-01

    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-κB and decreased intracellular level of its inhibitor IkBα. These effects, accompanied by increased production of H 2 O 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-κB 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

  1. The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

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    Simon Auer

    2017-05-01

    Full Text Available NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase, where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

  2. ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Drummond, Heather A

    2011-01-01

    The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. Copyright © 2011 S. Karger AG, Basel.

  3. Low-Dose Dextromethorphan, a NADPH Oxidase Inhibitor, Reduces Blood Pressure and Enhances Vascular Protection in Experimental Hypertension

    Science.gov (United States)

    Wu, Tao-Cheng; Chao, Chih-Yu; Lin, Shing-Jong; Chen, Jaw-Wen

    2012-01-01

    Background Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs). Methodology/Principal Findings Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. Conclusions/Significance Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension. PMID:23049937

  4. Low-dose dextromethorphan, a NADPH oxidase inhibitor, reduces blood pressure and enhances vascular protection in experimental hypertension.

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    Tao-Cheng Wu

    Full Text Available BACKGROUND: Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM, a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP and vascular protection in aged spontaneous hypertensive rats (SHRs. METHODOLOGY/PRINCIPAL FINDINGS: Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells. CONCLUSIONS/SIGNIFICANCE: Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension.

  5. NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity.

    Science.gov (United States)

    Gupta, D K; Pena, L B; Romero-Puertas, M C; Hernández, A; Inouhe, M; Sandalio, L M

    2017-04-01

    The role of NADPH oxidases under cadmium (Cd) toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF, which were grown under hydroponic conditions with 25 and 100 μM Cd for 1 and 5 days. Cadmium reduced the growth of leaves in WT, AtrbohC and D, but not in AtrbohF. A time-dependent increase in H 2 O 2 and lipid peroxidation was observed in all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was observed with NO accumulation. Cadmium increased catalase activity in WT plants and decreased it in Atrboh mutants, while glutathione reductase and glycolate oxidase activities increased in Atrboh mutants, and superoxide dismutases were down-regulated in AtrbohC. The GSH/GSSG and ASA/DHA couples were also affected by the treatment, principally in AtrbohC and AtrbohF, respectively. Cadmium translocation to the leaves was severely reduced in Atrboh mutants after 1 day of treatment and even after 5 days in AtrbohF. Similar results were observed for S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K accumulation, except in AtrbohF. Thus, under Cd stress, RBOHs differentially regulate ROS metabolism, redox homeostasis and nutrient balance and could be of potential interest in biotechnology for the phytoremediation of polluted soils. © 2016 John Wiley & Sons Ltd.

  6. NADPH promotes the rapid growth of the tumor

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    Hao Sheng

    2018-04-01

    Full Text Available NADPH oxidase is the main source of intracellular reactive oxygen species (ROS. ROS plays an important role in a variety of tumor types. The ROS mediated by NADPH oxidase increases the expression of hypoxia-inducible factor alpha (HIF-α through multiple signaling pathways in tumor, and HIF-α could be regulated and controlled by downstream multiple targeted genes such as vascular endothelial growth factor, glucose transporter to promote tumor angiogenesis, cell energy metabolism reprogram and tumor metastasis. Meanwhile, HIF-α can also regulate the expression of NADPH oxidase by ROS, thus further promoting development of tumor. In this review, we summarized the functions of NADPH in tumorigenesis and discussed their potential implications in cancer therapy.

  7. Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase.

    Science.gov (United States)

    Block, M L; Wu, X; Pei, Z; Li, G; Wang, T; Qin, L; Wilson, B; Yang, J; Hong, J S; Veronesi, B

    2004-10-01

    The contributing role of environmental factors to the development of Parkinson's disease has become increasingly evident. We report that mesencephalic neuron-glia cultures treated with diesel exhaust particles (DEP; 0.22 microM) (5-50 microg/ml) resulted in a dose-dependent decrease in dopaminergic (DA) neurons, as determined by DA-uptake assay and tyrosine-hydroxylase immunocytochemistry (ICC). The selective toxicity of DEP for DA neurons was demonstrated by the lack of DEP effect on both GABA uptake and Neu-N immunoreactive cell number. The critical role of microglia was demonstrated by the failure of neuron-enriched cultures to exhibit DEP-induced DA neurotoxicity, where DEP-induced DA neuron death was reinstated with the addition of microglia to neuron-enriched cultures. OX-42 ICC staining of DEP treated neuron-glia cultures revealed changes in microglia morphology indicative of activation. Intracellular reactive oxygen species and superoxide were produced from enriched-microglia cultures in response to DEP. Neuron-glia cultures from NADPH oxidase deficient (PHOX-/-) mice were insensitive to DEP neurotoxicity when compared with control mice (PHOX+/+). Cytochalasin D inhibited DEP-induced superoxide production in enriched-microglia cultures, implying that DEP must be phagocytized by microglia to produce superoxide. Together, these in vitro data indicate that DEP selectively damages DA neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult.

  8. Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

    OpenAIRE

    Gandara, Ana Caroline Paiva; Torres, Andr?; Bahia, Ana Cristina; Oliveira, Pedro L.; Schama, Renata

    2017-01-01

    Background NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not b...

  9. Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils

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    Gangling Chen

    2016-11-01

    Full Text Available Limb remote ischemic postconditioning (LRIP has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH oxidase and Myeloid differentiation factor 88 (MyD88-Tumor necrosis factor (TNF receptor-associated factor 6 (TRAF6-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345 in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.

  10. Chlorella induces stomatal closure via NADPH oxidase-dependent ROS production and its effects on instantaneous water use efficiency in Vicia faba.

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    Yan Li

    Full Text Available 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.

  11. Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    Science.gov (United States)

    Lin, Chih-Chung; Yang, Chien-Chung; Cho, Rou-Ling; Wang, Chen-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with

  12. Tea polyphenols alleviate high fat and high glucose-induced endothelial hyperpermeability by attenuating ROS production via NADPH oxidase pathway.

    Science.gov (United States)

    Zuo, Xuezhi; Tian, Chong; Zhao, Nana; Ren, Weiye; Meng, Yi; Jin, Xin; Zhang, Ying; Ding, Shibin; Ying, Chenjiang; Ye, Xiaolei

    2014-03-02

    Hyperglycemia-induced endothelial hyperpermeability is crucial to cardiovascular disorders and macro-vascular complications in diabetes mellitus. The objective of this study is to investigate the effects of green tea polyphenols (GTPs) on endothelial hyperpermeability and the role of nicotinamide adenine dinucleotide phosphate (NADPH) pathway. Male Wistar rats fed on a high fat diet (HF) were treated with GTPs (0, 0.8, 1.6, 3.2 g/L in drinking water) for 26 weeks. Bovine aortic endothelial cells (BAECs) were treated with high glucose (HG, 33 mmol/L) and GTPs (0.0, 0.4, or 4 μg/mL) for 24 hours in vitro. The endothelial permeabilities in rat aorta and monolayer BAECs were measured by Evans blue injection method and efflux of fluorescein isothiocyanate (FITC)-dextran, respectively. The reactive oxygen species (ROS) levels in rat aorta and monolayer BAECs were measured by dihydroethidium (DHE) and 2', 7'-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe, respectively. Protein levels of NADPH oxidase subunits were determined by Western-blot. HF diet-fed increased the endothelial permeability and ROS levels in rat aorta while HG treatments increased the endothelial permeability and ROS levels in cultured BAECs. Co-treatment with GTPs alleviated those changes both in vivo and in vitro. In in vitro studies, GTPs treatments protected against the HG-induced over-expressions of p22phox and p67phox. Diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase, alleviated the hyperpermeability induced by HG. GTPs could alleviate endothelial hyperpermeabilities in HF diet-fed rat aorta and in HG treated BAECs. The decrease of ROS production resulting from down-regulation of NADPH oxidase contributed to the alleviation of endothelial hyperpermeability.

  13. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    International Nuclear Information System (INIS)

    Riganti, Chiara; Costamagna, Costanzo; Bosia, Amalia; Ghigo, Dario

    2006-01-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H 2 O 2 concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution

  14. Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

    NARCIS (Netherlands)

    Weyemi, Urbain; Caillou, Bernard; Talbot, Monique; Ameziane-El-Hassani, Rabii; Lacroix, Ludovic; Lagent-Chevallier, Odile; Al Ghuzlan, Abir; Roos, Dirk; Bidart, Jean-Michel; Virion, Alain; Schlumberger, Martin; Dupuy, Corinne

    2010-01-01

    NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR,

  15. Apocynin: Chemical and Biophysical Properties of a NADPH Oxidase Inhibitor

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    Valdecir F. Ximenes

    2013-03-01

    Full Text Available Apocynin is the most employed inhibitor of NADPH oxidase (NOX, a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V, the hydrophobicity index was calculated (logP = 0.83 and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M−1 cm−1. Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA with a binding affinity of 2.19 × 104 M−1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

  16. Effects of co-administration of dietary sodium arsenite and an NADPH oxidase inhibitor on the rat bladder epithelium

    International Nuclear Information System (INIS)

    Suzuki, Shugo; Arnold, Lora L.; Pennington, Karen L.; Kakiuchi-Kiyota, Satoko; Cohen, Samuel M.

    2009-01-01

    Arsenite (As III ), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As III and dimethylarsinous acid (DMA III ) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as L-ascorbate and N-acetylcysteine, did not inhibit As III -induced cytotoxicity but they were more effective at inhibiting DMA III -induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100 ppm As III . Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As III treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As III -induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

  17. Zinc Oxide Nanoparticle Induces Microglial Death by NADPH-Oxidase-Independent Reactive Oxygen Species as well as Energy Depletion.

    Science.gov (United States)

    Sharma, Anuj Kumar; Singh, Vikas; Gera, Ruchi; Purohit, Mahaveer Prasad; Ghosh, Debabrata

    2017-10-01

    Zinc oxide nanoparticle (ZnO-NP) is one of the most widely used engineered nanoparticles. Upon exposure, nanoparticle can eventually reach the brain through various routes, interact with different brain cells, and alter their activity. Microglia is the fastest glial cell to respond to any toxic insult. Nanoparticle exposure can activate microglia and induce neuroinflammation. Simultaneous to activation, microglial death can exacerbate the scenario. Therefore, we focused on studying the effect of ZnO-NP on microglia and finding out the pathway involved in the microglial death. The present study showed that the 24 h inhibitory concentration 50 (IC 50 ) of ZnO-NP for microglia is 6.6 μg/ml. Early events following ZnO-NP exposure involved increase in intracellular calcium level as well as reactive oxygen species (ROS). Neither of NADPH oxidase inhibitors, apocynin, (APO) and diphenyleneiodonium chloride (DPIC) were able to reduce the ROS level and rescue microglia from ZnO-NP toxicity. In contrary, N-acetyl cysteine (NAC) showed opposite effect. Exogenous supplementation of superoxide dismutase (SOD) reduced ROS significantly even beyond control level but partially rescued microglial viability. Interestingly, pyruvate supplementation rescued microglia near to control level. Following 10 h of ZnO-NP exposure, intracellular ATP level was measured to be almost 50 % to the control. ZnO-NP-induced ROS as well as ATP depletion both disturbed mitochondrial membrane potential and subsequently triggered the apoptotic pathway. The level of apoptosis-inducing proteins was measured by western blot analysis and found to be upregulated. Taken together, we have deciphered that ZnO-NP induced microglial apoptosis by NADPH oxidase-independent ROS as well as ATP depletion.

  18. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    Science.gov (United States)

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  19. Activation of PAR-1/NADPH Oxidase/ROS Signaling Pathways is Crucial for the Thrombin-Induced sFlt-1 Production in Extravillous Trophoblasts: Possible Involvement in the Pathogenesis of Preeclampsia

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    Qi-tao Huang

    2015-03-01

    Full Text Available Backgrounds/Aims: Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1 expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT. Methods: An EVT cell line (HRT-8/SVneo was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS production were determined by DCFH-DA. Results: Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Conclusions: Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future.

  20. Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

    Science.gov (United States)

    Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

    2015-01-01

    Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

  1. Inhibitors of NADPH oxidase decrease endotoxin mediated induction of inducible nitric oxide expression in mouse macrophages

    Czech Academy of Sciences Publication Activity Database

    Krejčová, Daniela; Okénková, Kateřina; Konopka, Roman; Lojek, Antonín; Kubala, Lukáš

    2007-01-01

    Roč. 101, č. 14 (2007), s203-s204 E-ISSN 1213-7103. [Mezioborová česko-slovenská toxikologická konference /12./. Praha, 11.06.2007-13.06.2007] R&D Projects: GA ČR(CZ) GA524/06/1197 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lipopolysaccharide * inhibitors of NADPH oxidase * macrophage s Subject RIV: BO - Biophysics

  2. NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome.

    Science.gov (United States)

    Onetti, Yara; Meirelles, Thayna; Dantas, Ana P; Schröder, Katrin; Vila, Elisabet; Egea, Gustavo; Jiménez-Altayó, Francesc

    2016-05-01

    Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS. Copyright © 2016 the American Physiological Society.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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 D_2 (PGD_2) 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. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

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

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Shubin [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shimamoto, Shigeru [Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502 (Japan); Maruno, Takahiro; Kobayashi, Yuji [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kawahara, Kazuki; Yoshida, Takuya [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-12-04

    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 D{sub 2} (PGD{sub 2}) 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{sup +}, 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. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  5. TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.

    Science.gov (United States)

    Ma, Ming-Ming; Gao, Min; Guo, Kai-Min; Wang, Mi; Li, Xiang-Yu; Zeng, Xue-Lin; Sun, Lu; Lv, Xiao-Fei; Du, Yan-Hua; Wang, Guan-Lei; Zhou, Jia-Guo; Guan, Yong-Yuan

    2017-05-01

    Ca 2+ -activated Cl - channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca 2+ -activated Cl - channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases. © 2017 American Heart Association, Inc.

  6. Neuroprotection of taurine against reactive oxygen species is associated with inhibiting NADPH oxidases.

    Science.gov (United States)

    Han, Zhou; Gao, Li-Yan; Lin, Yu-Hui; Chang, Lei; Wu, Hai-Yin; Luo, Chun-Xia; Zhu, Dong-Ya

    2016-04-15

    It is well established that taurine shows potent protection against glutamate-induced injury to neurons in stroke. The neuroprotection may result from multiple mechanisms. Increasing evidences suggest that NADPH oxidases (Nox), the primary source of superoxide induced by N-methyl-d-aspartate (NMDA) receptor activation, are involved in the process of oxidative stress. We found that 100μM NMDA induced oxidative stress by increasing the reactive oxygen species level, which contributed to the cell death, in vitro. Neuron cultures pretreated with 25mM taurine showed lower percentage of death cells and declined reactive oxygen species level. Moreover, taurine attenuated Nox2/Nox4 protein expression and enzyme activity and declined intracellular calcium intensity during NMDA-induced neuron injury. Additionally, taurine also showed neuroprotection against H2O2-induced injury, accompanying with Nox inhibition. So, we suppose that protection of taurine against reactive oxygen species during NMDA-induced neuron injury is associated with Nox inhibition, probably in a calcium-dependent manner. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. QSYQ Attenuates Oxidative Stress and Apoptosis Induced Heart Remodeling Rats through Different Subtypes of NADPH-Oxidase

    Directory of Open Access Journals (Sweden)

    Yong Wang

    2013-01-01

    Full Text Available We aim to investigate the therapeutic effects of QSYQ, a drug of heart failure (HF in clinical practice in China, on a rat heart failure (HF model. 3 groups were divided: HF model group (LAD ligation, QSYQ group (LAD ligation and treated with QSYQ, and sham-operated group. After 4 weeks, rats were sacrificed for cardiac injury measurements. Rats with HF showed obvious histological changes including necrosis and inflammation foci, elevated ventricular remodeling markers levels(matrix metalloproteinases-2, MMP-2, deregulated ejection fraction (EF value, increased formation of oxidative stress (Malondialdehyde, MDA, and up-regulated levels of apoptotic cells (caspase-3, p53 and tunnel in myocardial tissue. Treatment of QSYQ improved cardiac remodeling through counter-acting those events. The improvement of QSYQ was accompanied with a restoration of NADPH oxidase 4 (NOX4 and NADPH oxidase 2 (NOX2 pathways in different patterns. Administration of QSYQ could attenuate LAD-induced HF, and AngII-NOX2-ROS-MMPs pathway seemed to be the critical potential targets for QSYQ to reduce the remodeling. Moreover, NOX4 was another key targets to inhibit the p53 and Caspase3, thus to reduce the hypertrophy and apoptosis, and eventually provide a synergetic cardiac protective effect.

  8. Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

    Science.gov (United States)

    Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 ·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 ·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 ·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 ·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 ·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

  9. Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome.

    Directory of Open Access Journals (Sweden)

    Victoria Campuzano

    2012-02-01

    Full Text Available A hallmark feature of Williams-Beuren Syndrome (WBS is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX-mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII, oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII-mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism.

  10. Identification of the NADPH Oxidase 4 Inhibiting Principle of Lycopus europaeus

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    Silvia Revoltella

    2018-03-01

    Full Text Available NADPH oxidase 4 (Nox4 has recently been implicated as driving force in cellular senescence. Thus, there is growing interest to develop Nox4 inhibitors, which might be valuable agents for cosmeceutical applications. Alpine plants represent a valuable source for the identification of novel bioactive natural products with anti-ageing effects, especially substances that protect plants against UV radiation, which is also known to contribute to the ageing of human skin. Therefore, the aim of this study was to identify novel Nox4 inhibitors from alpine plants. Within an initial screening of extracts of alpine plants on their ability to inhibit Nox4 activity in HEK cells, the methanolic extract of the subaerial parts of Lycopus europaeus showed a strong inhibition of Nox4 (81% chemiluminescence quenching and a simultaneously high cell viability (91% vitality. Rosmarinic acid was isolated and identified as the major compound in this bioactive extract. It showed a dose dependent inhibitory activity on Nox4 with an IC50 of 1 µM. Moreover, it also showed a significant inhibitory activity on Nox2 in the low micromolar range, whereas no inhibition of Nox5 was detected. Further investigations confirmed that the observed effects of rosmarinic acid on Nox2 and Nox4 are real inhibitory activities, and not due to ROS scavenging effects. Therefore, L. europaeus, which we demonstrated to be a good source of rosmarinic acid, has great potential for usage in cosmeceutical products with anti-ageing activity.

  11. Leonurine (SCM-198) attenuates myocardial fibrotic response via inhibition of NADPH oxidase 4.

    Science.gov (United States)

    Liu, Xin-Hua; Pan, Li-Long; Deng, Hai-Yan; Xiong, Qing-Hui; Wu, Dan; Huang, Guo-Ying; Gong, Qi-Hai; Zhu, Yi-Zhun

    2013-01-01

    In our previous studies, we have reported that leonurine, a plant phenolic alkaloid in Herba leonuri, exerted cardioprotective properties in a number of preclinical experiments. Herein, we investigated the roles and the possible mechanisms of leonurine for reducing fibrotic responses in angiotensin II (Ang II)-stimulated primary neonatal rat cardiac fibroblasts and post-myocardial infarction (MI) rats. In in vitro experiments performed in neonatal rat cardiac fibroblasts, leonurine (10-20 μM) pretreatment attenuated Ang II-induced activation of extracellular signal-regulated kinase 1/2, production of intracellular reactive oxygen species (ROS), expression and activity of matrix metalloproteinase (MMP)-2/9, and expression of α-smooth muscle actin and types I and III collagen. A small interfering RNA-mediated knockdown strategy for NADPH oxidase 4 (Nox4) revealed that Nox4 was required for Ang II-induced activation of cardiac fibroblasts. In vivo studies using a post-MI model in rats indicated that administration of leonurine inhibited myocardial fibrosis while reducing cardiac Nox4 expression, ROS production, NF-κB activation, and plasma MMP-2 activity. In conclusion, our results provide the first evidence that leonurine could prevent cardiac fibrosis and the activation of cardiac fibroblasts partly through modulation of a Nox4-ROS pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

    Science.gov (United States)

    Wang, Su Bin; Jang, Ji Yong; Chae, Yun Hee; Min, Ji Hyun; Baek, Jin Young; Kim, Myunghee; Park, Yunjeong; Hwang, Gwi Seo; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-06-01

    Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation.

    Directory of Open Access Journals (Sweden)

    Christine Deffert

    2014-09-01

    Full Text Available Patients with chronic granulomatous disease (CGD lack generation of reactive oxygen species (ROS through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%. The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter. Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (∼ 50%. Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-α, IFN-γ, IL-17 and IL-12 early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine

  14. Sphingosine 1-phosphate-induced ICAM-1 expression via NADPH oxidase/ROS-dependent NF-kappaB cascade on human pulmonary alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Chin-Chung eLin

    2016-03-01

    Full Text Available The intercellular adhesion molecule-1 (ICAM-1 expression is frequently correlated with the lung inflammation. A bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P, was involved in inflammation through the adhesion molecules induction, and then caused lung injury. However, the transduction mechanisms of the S1P stimulation to induce ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs remain unclear. Here, we demonstrated that exposure of HPAEpiCs to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCdelta, PF431396 (PYK2, diphenyleneiodonium chloride (DPI, apocynin (NADPH oxidase, Edaravone (ROS, and Bay11-7082 (NF-kappaB. Consistently, knockdown with siRNA transfection of PKCdelta, PYK2, p47phox, and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A and Gi/o-coupled receptor antagonist (GPA2 also blocked S1P-induced ICAM-1 protein and mRNA expression. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCdelta-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-kappaB p65 phosphorylation and translocation from the cytosol to the nucleus in HPAEpiCs, which was inhibited by Rottlerin, PF431396, APO, DPI, or Edaravone. In the in vitro study, we established that S1P induced monocyte adhesion via an ICAM-1-dependent pathway. In the in vivo study, we found that S1P induced ICAM-1 protein and mRNA levels in the lung fractions, pulmonary hematoma, and leukocyte (mainly eosinophils and neutrophils count in bronchoalveolar lavage (BAL fluid in mice via a PKCdelta/PYK2/NADPH oxidase/ROS/NF-kappaB signaling pathway. We concluded that S1P may induce lung

  15. Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes

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    Simona-Adriana Manea

    2018-06-01

    Full Text Available Reactive oxygen species (ROS generated by up-regulated NADPH oxidase (Nox contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA, a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression. Keywords: NADPH oxidase, Epigenetics, HDAC, Histone acetylation, Diabetes

  16. Andrographolide inhibits TNFα-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.

    Science.gov (United States)

    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 TNFα-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 TNFα-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 TNFα-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. Copyright © 2014. Published by Elsevier Inc.

  17. Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

    Science.gov (United States)

    Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David

    2012-06-22

    NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. The role of oxidative stress and NADPH oxidase in the pathogenesis of atherosclerosis

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    Dorota Bryk

    2017-01-01

    Full Text Available Reactive oxygen species (ROS play a key role in the pathogenesis of atherosclerosis. The main mechanisms which are involved are low-density lipoprotein oxidative modification, inactivation of nitric oxide and modulation of redox-sensitive signaling pathways. ROS contribute to several aspects of atherosclerosis including endothelial cell dysfunction, monocyte/macrophage recruitment and activation, stimulation of inflammation, and inducing smooth muscle cell migration and proliferation. NADPH oxidase is the main source of ROS in the vasculature. This enzyme consists of a membrane-bound heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox, p67phox and p40phox, and small GTP-binding proteins rac1 and rac 2. Seven distinct isoforms of this enzyme have been identified, of which four (NOX1, 2, 4 and 5 may have cardiovascular function. In this paper, we review the current state of knowledge concerning the role of oxidative stress and NOX enzymes in pathogenesis of atherosclerosis. Moreover, we analyze the experimental studies that explore the relationship between the NOX family and atherosclerosis.

  19. Isolation of a cotton NADP(H oxidase homologue induced by drought stress

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    NEPOMUCENO ALEXANDRE LIMA

    2000-01-01

    Full Text Available The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L. genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

  20. Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Hood, Katie Y; Mair, Kirsty M; Harvey, Adam P; Montezano, Augusto C; Touyz, Rhian M; MacLean, Margaret R

    2017-07-01

    Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1 -/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT 1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT 1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT 1B receptor signaling and Nox1, confirmed in PASMCs from Nox1 -/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT 1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT 1B receptors contribute to

  1. Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

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    Yoo, T; Ham, S A; Hwang, J S; Lee, W J; Paek, K S; Oh, J W; Kim, J H; Do, J T; Han, C W; Kim, J H; Seo, H G

    2016-10-01

    We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Associations Between Genetic Variants of NADPH Oxidase-Related Genes and Blood Pressure Responses to Dietary Sodium Intervention: The GenSalt Study.

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    Han, Xikun; Hu, Zunsong; Chen, Jing; Huang, Jianfeng; Huang, Chen; Liu, Fangchao; Gu, Charles; Yang, Xueli; Hixson, James E; Lu, Xiangfeng; Wang, Laiyuan; Liu, De-Pei; He, Jiang; Chen, Shufeng; Gu, Dongfeng

    2017-04-01

    The aim of this study was to comprehensively test the associations of genetic variants of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-related genes with blood pressure (BP) responses to dietary sodium intervention in a Chinese population. We conducted a 7-day low-sodium intervention followed by a 7-day high-sodium intervention among 1,906 participants in rural China. BP measurements were obtained at baseline and each dietary intervention using a random-zero sphygmomanometer. Linear mixed-effect models were used to assess the additive associations of 63 tag single-nucleotide polymorphisms in 11 NADPH oxidase-related genes with BP responses to dietary sodium intervention. Gene-based analyses were conducted using the truncated product method. The Bonferroni method was used to adjust for multiple testing in all analyses. Systolic BP (SBP) response to high-sodium intervention significantly decreased with the number of minor T allele of marker rs6967221 in RAC1 (P = 4.51 × 10-4). SBP responses (95% confidence interval) for genotypes CC, CT, and TT were 5.03 (4.71, 5.36), 4.20 (3.54, 4.85), and 0.56 (-1.08, 2.20) mm Hg, respectively, during the high-sodium intervention. Gene-based analyses revealed that RAC1 was significantly associated with SBP response to high-sodium intervention (P = 1.00 × 10-6) and diastolic BP response to low-sodium intervention (P = 9.80 × 10-4). These findings suggested that genetic variants of NADPH oxidase-related genes may contribute to the variation of BP responses to sodium intervention in Chinese population. Further replication of these findings is warranted. © American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  3. Decoding NADPH oxidase 4 expression in human tumors

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    Jennifer L. Meitzler

    2017-10-01

    Full Text Available NADPH oxidase 4 (NOX4 is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients, esophagus (12/18 patients, bladder (10/19 patients, ovary (6/17 patients, and prostate (7/19 patients, as well as malignant melanoma (7/15 patients when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

  4. Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury.

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    Bo Young Jeong

    Full Text Available Contrast-induced acute kidney injury (CIAKI is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH oxidases (Noxs are important sources of reactive oxygen species (ROS. Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831. Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38 implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2'-deoxyguanosine (8OHdG and kidney injury molecule-1 (KIM-1, and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

  5. Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

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    Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

    2018-01-01

    Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

  6. Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

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    Meraj A. Khan

    2018-02-01

    Full Text Available Neutrophils migrating from the blood (pH 7.35–7.45 into the surrounding tissues encounter changes in extracellular pH (pHe conditions. Upon activation of NADPH oxidase 2 (Nox, neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi. Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET formation (NETosis is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements. Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs. In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative-, and Staphylococcus aureus (Gram-positive-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

  7. Association between NADPH oxidase p22(phox C242T polymorphism and ischemic cerebrovascular disease: a meta-analysis.

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    Bing-Hu Li

    Full Text Available BACKGROUND: Epidemiological studies have evaluated the association between nicotinamide adenine dinucleotide phosphate (NADPH oxidase p22(phox C242T polymorphism and risk of ischemic cerebrovascular disease (ICVD, but the results remain inconclusive. This meta-analysis was therefore designed to clarify these controversies. METHODOLOGY/PRINCIPAL FINDINGS: Systematic searches of electronic databases Embase, PubMed and Web of Science, as well as hand searching of the references of identified articles and the meeting abstracts were performed. Statistical analyses were performed using software Review Manager (Version 5.1.7 and Stata (Version 11.0. The pooled odds ratios (ORs with 95% confidence intervals (95%CIs were performed. Fixed or random effects model was separately used depending on the heterogeneity between studies. Publication bias was tested by Begg's funnel plot and Egger's regression test. A total of 6 studies including 1,948 cases and 2,357 controls were combined showing no statistical evidence of association between NADPH oxidase p22(phox C242T polymorphism and overall ICVD (allelic model: OR = 1.08, 95%CI = 0.93-1.26; additive model: OR = 1.33, 95%CI = 0.81-2.17; dominant model: OR = 1.00, 95%CI = 0.86-1.15; recessive model: OR = 1.06, 95%CI = 0.77-1.45. Significant association was found in large-artery atherosclerotic stroke subgroup (allelic model: OR = 1.12, 95%CI = 0.88-1.41; additive model: OR = 1.36, 95%CI = 0.60-3.09; dominant model: OR = 1.25, 95%CI = 0.74-2.11; recessive model: OR = 2.17, 95%CI = 1.11-4.23. No statistical evidence of significant association was observed for small-vessel occlusive stroke, as well as Asian subgroup and Caucasian subgroup. Statistical powers on the combined sample size (total and subgroup were all lower than 80%. CONCLUSIONS/SIGNIFICANCE: This meta-analysis indicates that NADPH oxidase p22(phox C242T polymorphism is more associated

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  9. Glucose impairs aspirin inhibition in platelets through a NAD(P)H oxidase signaling pathway.

    Science.gov (United States)

    Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

    2017-07-01

    Hyperglycemia has been suggested to play a role in the increased platelet resistance to antiplatelet therapy in patients with diabetes mellitus. Exposure to high glucose impairs platelet inhibition by aspirin. It has been found that antioxidant agents reduce the effect of glucose, confirming the involvement of reactive oxygen species (ROS) in the effect of glucose. The aim of the study was to examine the mechanism of ROS increase by high glucose in aspirin-treated platelets. Platelet aggregation was measured by the optical method, and the production of ROS was detected using luminol-dependent horseradish peroxidase-enhanced chemiluminescence. We found that glucose did not affect ADP-induced platelet aggregation. However, it reduced the effect of aspirin on platelet aggregation, which was accompanied by an increase in ROS generation. The inhibition of NAD(P)H oxidase (NOX) prevented the glucose effect and ROS generation. The same result was recorded after the inhibition of p38 mitogen-activated protein kinases (p38 MAPK), phospholipase A 2 (PLA 2 ) or 12-lipoxygenase (12-LOX). The inhibition of TxA 2 receptor did not decrease the effect of glucose indicating that the effect was not caused by activation of TxA 2 receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase- and COX-derived oxidative stress.

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    Niazi, Zahid Rasul; Silva, Grazielle C; Ribeiro, Thais Porto; León-González, Antonio J; Kassem, Mohamad; Mirajkar, Abdur; Alvi, Azhar; Abbas, Malak; Zgheel, Faraj; Schini-Kerth, Valérie B; Auger, Cyril

    2017-12-01

    Eicosapentaenoic acid:docosahexaenoic acid (EPA:DHA) 6:1, an omega-3 polyunsaturated fatty acid formulation, has been shown to induce a sustained formation of endothelial nitric oxide (NO) synthase-derived NO, a major vasoprotective factor. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Male Wister rats received orally corn oil or EPA:DHA 6:1 (500 mg kg -1 per day) before chronic infusion of Ang II (0.4 mg kg -1 per day). Systolic blood pressure was determined by tail cuff sphingomanometry, vascular reactivity using a myograph, oxidative stress using dihydroethidium and protein expression by immunofluorescence and western blot analysis. Ang II-induced hypertension was associated with reduced acetylcholine-induced relaxations of secondary branch mesenteric artery rings affecting the endothelium-dependent hyperpolarization (EDH)- and the NO-mediated relaxations, both of which were improved by the NADPH oxidase inhibitor VAS-2870. The Ang II treatment induced also endothelium-dependent contractile responses (EDCFs), which were abolished by the cyclooxygenase (COX) inhibitor indomethacin. An increased level of vascular oxidative stress and expression of NADPH oxidase subunits (p47 phox and p22 phox ), COX-1 and COX-2, endothelial NO synthase and Ang II type 1 receptors were observed in the Ang II group, whereas SK Ca and connexin 37 were downregulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction by improving both the NO- and EDH-mediated relaxations, and by reducing EDCFs and the expression of target proteins. The present findings indicate that chronic intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidase- and COX-derived oxidative stress.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  13. Allelic variations in the CYBA gene of NADPH oxidase and risk of kidney complications in patients with type 1 diabetes.

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    Patente, Thiago A; Mohammedi, Kamel; Bellili-Muñoz, Naïma; Driss, Fathi; Sanchez, Manuel; Fumeron, Frédéric; Roussel, Ronan; Hadjadj, Samy; Corrêa-Giannella, Maria Lúcia; Marre, Michel; Velho, Gilberto

    2015-09-01

    Oxidative stress plays a pivotal role in the pathophysiology of diabetic nephropathy, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is an important source of reactive oxygen species in hyperglycemic conditions in the kidney. Plasma concentration of advanced oxidation protein products (AOPP), a marker of oxidative stress, is increased in patients with diabetic nephropathy. We investigated associations of variants in the CYBA gene, encoding the regulatory subunit p22(phox) of NADPH oxidase, with diabetic nephropathy and plasma AOPP and myeloperoxidase (MPO) concentrations in type 1 diabetic patients. Seven SNPs in the CYBA region were analyzed in 1357 Caucasian subjects with type 1 diabetes from the SURGENE (n=340), GENEDIAB (n=444), and GENESIS (n=573) cohorts. Duration of follow-up was 10, 9, and 6 years, respectively. Cox proportional hazards and logistic regression analyses were used to estimate hazard ratios (HR) or odds ratios (OR) for incidence and prevalence of diabetic nephropathy. The major G-allele of rs9932581 was associated with the incidence of renal events defined as new cases of microalbuminuria or the progression to a more severe stage of nephropathy during follow-up (HR 1.59, 95% CI 1.17-2.18, P=0.003) in SURGENE. The same allele was associated with established/advanced nephropathy (OR 1.52, 95% CI 1.22-1.92, P=0.0001) and with the incidence of end-stage renal disease (ESRD) (HR 2.01, 95% CI 1.30-3.24, P=0.001) in GENEDIAB/GENESIS pooled studies. The risk allele was also associated with higher plasma AOPP concentration in subsets of SURGENE and GENEDIAB, with higher plasma MPO concentration in a subset of GENEDIAB, and with lower estimated glomerular filtration rate (eGFR) in the three cohorts. In conclusion, a functional variant in the promoter of the CYBA gene was associated with lower eGFR and with prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. These results are consistent with

  14. Calcineurin Aβ regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.

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    Williams, Clintoria R; Gooch, Jennifer L

    2014-02-21

    Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aβ is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα(-/-) or CnAβ(-/-)), we found that high glucose selectively activates CnAβ, whereas CnAα is constitutively active. Furthermore, CnAβ but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAβ(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAβ reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAβ-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAβ/NFAT pathway modulates Nox. These data reveal that the CnAβ/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.

  15. Squamosamide derivative FLZ protects dopaminergic neurons against inflammation-mediated neurodegeneration through the inhibition of NADPH oxidase activity

    Directory of Open Access Journals (Sweden)

    Wilson Belinda

    2008-05-01

    Full Text Available Abstract Background Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases. Methods For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS- and 1-methyl-4-phenylpyridinium-(MPP+-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP- induced PD mouse model was used. Results FLZ showed potent efficacy in protecting dopaminergic (DA neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α, nitric oxide (NO and prostaglandin E2 (PGE2. Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX, the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1 FLZ's protective effect was reduced in cultures from PHOX-/- mice, and 2 FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal

  16. JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

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    Ling Yan

    2013-01-01

    Full Text Available Excessive fluoride may cause central nervous system (CNS dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS, and NADPH oxidase (NOX is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells.

  17. JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

    Science.gov (United States)

    Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

    2013-01-01

    Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

  18. The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase

    DEFF Research Database (Denmark)

    Holdfeldt, Andre; Skovbakke, Sarah Line; Winther, Malene

    2016-01-01

    Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists......2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated......2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases....

  19. Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation.

    Science.gov (United States)

    Kaneko, Chihiro; Ogura, Jiro; Sasaki, Shunichi; Okamoto, Keisuke; Kobayashi, Masaki; Kuwayama, Kaori; Narumi, Katsuya; Iseki, Ken

    2017-03-01

    A high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. We used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. Single administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. Single administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. The Role of NADPH Oxidase in the Inhibition of Trichophyton rubrum by 420-nm Intense Pulsed Light

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    Hao Huang

    2018-01-01

    Full Text Available Objectives: To evaluate the effect of intense pulsed light (IPL on Trichophyton rubrum and investigate its mechanism of action.Methods: The viability of fungi treated with IPL alone and with IPL combined with an NADPH oxidase inhibitor (DPI pretreatment was determined by MTT assays. The reactive oxygen species (ROS were quantified with a DCFH-DA fluorescent probe. Malondialdehyde (MDA content and superoxide dismutase (SOD and glutathione peroxidase (GSH-Px activities were determined by commercial kits. The transcription of the Nox gene was quantified using quantitative real-time PCR (qRT-PCR analysis, and micromorphology was observed using scanning electron microscopy (SEM. In addition, fungal keratinase activity was detected by measuring dye release from keratin azure.Results: The growth declined with statistical significance after 6 h of treatment (P < 0.001. The ROS and MDA content increased after IPL treatment, whereas the SOD and GSH-Px activity decreased. Nox gene expression was upregulated, and the micromorphology was damaged. Keratinase activity decreased. Fungi that received DPI pretreatment exhibited contrasting outcomes.Conclusion: We found that 420-nm IPL significantly inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism involves Nox as a factor that mediates 420-nm IPL-induced oxidative damage of T. rubrum.

  1. Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia

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    Ruoting Ding

    2016-12-01

    Full Text Available Oxidative stress is a possible pathogenesis of hyperalgesia. Advanced oxidation protein products (AOPPs, a new family of oxidized protein compounds, have been considered as a novel marker of oxidative stress. However, the role of AOPPs in the mechanism of hyperalgesia remains unknown. Our study aims to investigate whether AOPPs have an effect on hyperalgesia and the possible underlying mechanisms. To identify the AOPPs involved, we induced hyperalgesia in rats by injecting complete Freund’s adjuvant (CFA in hindpaw. The level of plasma AOPPs in CFA-induced rats was 1.6-fold in comparison with what in normal rats (P<0.05. After intravenous injection of AOPPs-modified rat serum albumin (AOPPs-RSA in Sprague-Dawley rats, the paw mechanical thresholds, measured by the electronic von Frey system, significantly declined. Immunofluorescence staining indicated that AOPPs increased expressions of NADPH oxidase 1 (Nox1, NADPH oxidase 4 (Nox4, transient receptor potential vanilloid 1 (TRPV1 and calcitonin gene-related peptide (CGRP in the dorsal root ganglia (DRG tissues. In-vitro studies were performed on primary DRG neurons which were obtained from both thoracic and lumbar DRG of rats. Results indicated that AOPPs triggered reactive oxygen species (ROS production in DRG neurons, which were significantly abolished by ROS scavenger N-acetyl-l-cysteine (NAC and small-interfering RNA (siRNA silencing of Nox1 or Nox4. The expressions of Nox1, Nox4, TRPV1 and CGRP were significantly increased in AOPPs-induced DRG neurons. And relevant siRNA or inhibitors notably suppressed the expressions of these proteins and the calcium influxes in AOPPs-induced DRG neurons. In conclusion, AOPPs increased significantly in CFA-induced hyperalgesia rats and they activated Nox1/Nox4-ROS to sensitize TRPV1-dependent Ca2+ influx and CGRP release which led to inducing mechanical hyperalgesia.

  2. Chronic granulomatous disease caused by mutations other than the common GT deletion in NCF1, the gene encoding the p47phox component of the phagocyte NADPH oxidase

    NARCIS (Netherlands)

    Roos, Dirk; de Boer, Martin; Köker, M. Yavuz; Dekker, Jan; Singh-Gupta, Vinita; Ahlin, Anders; Palmblad, Jan; Sanal, Ozden; Kurenko-Deptuch, Magdalena; Jolles, Stephen; Wolach, Baruch

    2006-01-01

    Chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by defects in any of four genes encoding components of the leukocyte nicotinamide dinucleotide phosphate, reduced (NADPH) oxidase. One of these is the autosomal neutrophil cytosolic factor 1 (NCF1) gene encoding the p47phox

  3. Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.

    Science.gov (United States)

    Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A

    2013-12-01

    Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an

  4. Implication of NADPH Oxidases in the Early Inflammation Process Generated by Cystic Fibrosis Cells

    Science.gov (United States)

    Pongnimitprasert, Nushjira; Hurtado, Margarita; Lamari, Foudil; El Benna, Jamel; Dupuy, Corinne; Fay, Michèle; Foglietti, Marie-José; Bernard, Maguy; Gougerot-Pocidalo, Marie-Anne; Braut-Boucher, Françoise

    2012-01-01

    In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality. The aim of this study was to further investigate whether oxidative stress could be involved in the early inflammatory process associated with CF pathogenesis. We used a model of CFTR defective epithelial cell line (IB3-1) and its reconstituted CFTR control (S9) cell line cultured in various ionic conditions. This study showed that IB3-1 and S9 cells expressed the NADPH oxidases (NOXs) DUOX1/2 and NOX2 at the same level. Nevertheless, several parameters participating in oxidative stress (increased ROS production and apoptosis, decreased total thiol content) were observed in IB3-1 cells cultured in hypertonic environment as compared to S9 cells and were inhibited by diphenyleneiodonium (DPI), a well-known inhibitor of NOXs; besides, increased production of the proinflammatory cytokines IL-6 and IL-8 by IB3-1 cells was also inhibited by DPI as compared to S9 cells. Furthermore, calcium ionophore (A23187), which upregulates DUOX and NOX2 activities, strongly induced oxidative stress and IL-8 and IL-6 overexpression in IB3-1 cells. All these events were suppressed by DPI, supporting the involvement of NOXs in the oxidative stress, which can upregulate proinflammatory cytokine production by the airway CFTR-deficient cells and trigger early pulmonary inflammation in CF patients. PMID:24049649

  5. Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

    Science.gov (United States)

    Batchuluun, Battsetseg; Inoguchi, Toyoshi; Sonoda, Noriyuki; Sasaki, Shuji; Inoue, Tomoaki; Fujimura, Yoshinori; Miura, Daisuke; Takayanagi, Ryoichi

    2014-01-01

    Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. Swell activated chloride channel function in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

    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.

  7. Convergent evolution of morphogenetic processes in fungi: Role of tetraspanins and NADPH oxidases 2 in plant pathogens and saprobes.

    Science.gov (United States)

    Malagnac, Fabienne; Bidard, Frédérique; Lalucque, Hervé; Brun, Sylvain; Lambou, Karine; Lebrun, Marc-Henri; Silar, Philippe

    2008-01-01

    Convergent evolution of trophic life style and morphological characters are very common in the fungal kingdom. Recently, we have shown that the same molecular machinery containing a tetraspanin and a NADPH oxidase has been recruited in two different fungal species for the same purpose (exiting from a melanized re-enforced cell at a focal weakened point), but at different stages of their development (ascospore germination and appressorium mediated penetration). Although this molecular machinery is required at these key developmental steps, it is also likely involved in specialized cellular functions at other stages of fungal development, as shown here for nutrient acquisition by Podospora anserina.

  8. Prolonged Exposure of Cortical Neurons to Oligomeric Amyloid-β Impairs NMDA Receptor Function Via NADPH Oxidase-Mediated ROS Production: Protective Effect of Green Tea (--Epigallocatechin-3-Gallate

    Directory of Open Access Journals (Sweden)

    Yan He

    2011-01-01

    Full Text Available Excessive production of Aβ (amyloid β-peptide has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease. Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species in neurons through an NMDA (N-methyl-D-aspartate-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III-tetrakis(4-benzoic acid-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pretreatment of neurons with EGCG [(–-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.

  9. NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

    Science.gov (United States)

    Deliyanti, Devy; Rana, Indrajeetsinh; Miller, Antonia G.; Agrotis, Alex; Armani, Roksana; Szyndralewiez, Cédric; Wingler, Kirstin; Touyz, Rhian M.; Cooper, Mark E.; Jandeleit-Dahm, Karin A.; Schmidt, Harald H.H.W.

    2014-01-01

    Abstract Aims: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Results: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Innovation: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Conclusions: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies. Antioxid. Redox Signal. 20, 2726–2740. PMID:24053718

  10. NADPH Oxidase 1 Modulates WNT and NOTCH1 Signaling To Control the Fate of Proliferative Progenitor Cells in the Colon▿

    Science.gov (United States)

    Coant, Nicolas; Ben Mkaddem, Sanae; Pedruzzi, Eric; Guichard, Cécile; Tréton, Xavier; Ducroc, Robert; Freund, Jean-Noel; Cazals-Hatem, Dominique; Bouhnik, Yoram; Woerther, Paul-Louis; Skurnik, David; Grodet, Alain; Fay, Michèle; Biard, Denis; Lesuffleur, Thécla; Deffert, Christine; Moreau, Richard; Groyer, André; Krause, Karl-Heinz; Daniel, Fanny; Ogier-Denis, Eric

    2010-01-01

    The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/β-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/β-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/β-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/β-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector β-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/β-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation. PMID:20351171

  11. 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▿†

    Science.gov (United States)

    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

  12. NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro.

    Directory of Open Access Journals (Sweden)

    Dan Meng

    Full Text Available Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4 in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs. Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.

  13. Effects of stevia on synaptic plasticity and NADPH oxidase level of CNS in conditions of metabolic disorders caused by fructose.

    Science.gov (United States)

    Chavushyan, V A; Simonyan, K V; Simonyan, R M; Isoyan, A S; Simonyan, G M; Babakhanyan, M A; Hovhannisyian, L E; Nahapetyan, Kh H; Avetisyan, L G; Simonyan, M A

    2017-12-19

    Excess dietary fructose intake associated with metabolic syndrome and insulin resistance and increased risk of developing type 2 diabetes. Previous animal studies have reported that diabetic animals have significantly impaired behavioural and cognitive functions, pathological synaptic function and impaired expression of glutamate receptors. Correction of the antioxidant status of laboratory rodents largely prevents the development of fructose-induced plurimetabolic changes in the nervous system. We suggest a novel concept of efficiency of Stevia leaves for treatment of central diabetic neuropathy. By in vivo extracellular studies induced spike activity of hippocampal neurons during high frequency stimulation of entorhinal cortex, as well as neurons of basolateral amygdala to high-frequency stimulation of the hippocampus effects of Stevia rebaudiana Bertoni plant evaluated in synaptic activity in the brain of fructose-enriched diet rats. In the conditions of metabolic disorders caused by fructose, antioxidant activity of Stevia rebaudiana was assessed by measuring the NOX activity of the hippocampus, amygdala and spinal cord. In this study, the characteristic features of the metabolic effects of dietary fructose on synaptic plasticity in hippocampal neurons and basolateral amygdala and the state of the NADPH oxidase (NOX) oxidative system of these brain formations are revealed, as well as the prospects for development of multitarget and polyfunctional phytopreparations (with adaptogenic, antioxidant, antidiabetic, nootropic activity) from native raw material of Stevia rebaudiana. Stevia modulates degree of expressiveness of potentiation/depression (approaches but fails to achieve the norm) by shifting the percentage balance in favor of depressor type of responses during high-frequency stimulation, indicating its adaptogenic role in plasticity of neural networks. Under the action of fructose an increase (3-5 times) in specific quantity of total fraction of NOX

  14. NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment.

    Directory of Open Access Journals (Sweden)

    Meghan A Jendrysik

    Full Text Available NADPH oxidase-2 (Nox2/gp91(phox and p47(phox deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/--ROS deficient mouse dendritic cells (DC secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/- DC bias more ovalbumin-specific CD4(+ T lymphocytes toward a Th1 phenotype than wild type (WT DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2O(2 attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/- DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.

  15. Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi.

    Science.gov (United States)

    Lacaze, Isabelle; Lalucque, Hervé; Siegmund, Ulrike; Silar, Philippe; Brun, Sylvain

    2015-03-01

    NADPH oxidases (Nox) are membrane complexes that produce O2(-). Researches in mammals, plants and fungi highlight the involvement of Nox-generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91(phox)/Nox2 is associated with p22(phox) forming the flavocytochrome b558 ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22(phox) gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22(phox) homologue as being mutated in the Podospora anserina mutant IDC(509). Functional studies show that the fungal p22(phox), PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91(phox) homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co-localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system. © 2014 John Wiley & Sons Ltd.

  16. Sistema NADPH oxidasa: nuevos retos y perspectivas = NADPH oxidase system: new challenges and perspectives

    Directory of Open Access Journals (Sweden)

    Arango Rincón, Julián Camilo

    2010-12-01

    Full Text Available El sistema NADPH oxidasa es un complejo multiproteico encargado de producir especies reactivas del oxígeno (ROS, por reactive oxygen species en diferentes células y tejidos. Es de gran importancia en las células fagocíticas (principalmente neutrófilos y macrófagos porque participa en la destrucción de microorganismos patógenos, mediante la fagocitosis y la formación de las trampas extracelulares de neutrófilos (NET, por neutrophils extracelular traps, así como en la activación de procesos inflamatorios. Las alteraciones en la producción de ROS por parte de las células fagocíticas a causa de defectos genéticos en los componentes del sistema generan la inmunodeficiencia primaria denominada enfermedad granulomatosa crónica (EGC. Este es un artículo de revisión sobre los componentes del sistema NADPH oxidasa, su distribución celular, mecanismo de activación y acción, así como de las funciones que desempeña en otros tejidos. Además, se revisan los defectos moleculares que llevan a la EGC y el tratamiento de esta, incluyendo la terapia con IFNγ, y finalmente las perspectivas para el estudio del sistema.

  17. Acupuncture elicits neuroprotective effect by inhibiting NAPDH oxidase-mediated reactive oxygen species production in cerebral ischaemia.

    Science.gov (United States)

    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

    2015-12-10

    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.

  18. The NADPH-oxidase AtRbohI plays a positive role in drought-stress response in Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    He, Huan [Nanjing Agricultural Univ. (China); Yan, Jingwei [Nanjing Agricultural Univ. (China); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Yu, Xiaoyun [Nanjing Agricultural Univ. (China); Liang, Yan [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Fang, Lin [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Scheller, Henrik Vibe [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Zhang, Aying [Nanjing Agricultural Univ. (China)

    2017-05-27

    As the major resource of reactive oxygen species (ROS), the NADPH oxidases (Rbohs) have been shown to play important roles in plant cells under normal growth and stress conditions. Although many family members of Rbohs were studied, little is known about the function of RbohI in Arabidopsis thaliana. Here, we report that exogenous ABA application decreases RbohI expression and mannitol significantly increases RbohI expression at transcript level. The RbohI transcripts were strongly detected in dry seeds and roots. The loss-of-function mutant rbohI exhibited sensitivity to ABA and mannitol stress during germination. Furthermore, the lateral root growth of rbohI was severely inhibited after treatment with mannitol stress. Overexpression of RbohI in Arabidopsis significantly improves the drought tolerance. Moreover, more H2O2 accumulated in RbohI overexpressors than in wild type plants in response to mannitol stress. Our conclusion is that AtRbohI functions in drought-stress response in Arabidopsis thaliana.

  19. Involvement of NADPH oxidase isoforms in the production of O2- manipulated by ABA in the senescing leaves of early-senescence-leaf (esl) mutant rice (Oryza sativa).

    Science.gov (United States)

    Li, Zhaowei; Wang, Fubiao; Zhao, Qian; Liu, Jianchao; Cheng, Fangmin

    2018-01-01

    In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (esl) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (Oryza sativa). The temporal expression levels of OsNox isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the esl mutant generated more O2- concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O2- generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O2- generation. The esl mutant exhibited significantly higher expression of OsNox2, OsNox5, OsNox6, and OsNox7 in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of OsNox1, OsNox3, and OsFR07 in the flag leaf of the esl mutant were significantly lower than those in the wild-type cultivar. The expression levels of OsNox2, OsNox5, OsNox6, and OsNox7 were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of OsNox2 and OsNox6 were dependent on the duration of ABA treatment. The inducible expression levels of OsNox5 and OsNox7 were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of OsNox1, OsNox3, and OsFR07 in the detached leaf segments. Therefore, OsNox2, OsNox5, OsNox6, and OsNox7 were probably involved in the ABA-induced O2- generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the

  20. 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

    Science.gov (United States)

    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.

  1. Association of a variant in the regulatory region of NADPH oxidase 4 gene and metabolic syndrome in patients with chronic hepatitis C.

    Science.gov (United States)

    Siqueira, Erika Rabelo Forte de; Pereira, Luciano Beltrao; Stefano, Jose Tadeu; Patente, Thiago; Cavaleiro, Ana Mercedes; Silva Vasconcelos, Luydson Richardson; Carmo, Rodrigo Feliciano; Moreira Beltrao Pereira, Leila Maria; Carrilho, Flair Jose; Corrêa-Giannella, Maria Lucia; Oliveira, Claudia P

    2015-03-28

    Given the important contribution of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system to the generation of reactive oxygen species induced by hepatitis C virus (HCV), we investigated two single nucleotide polymorphisms (SNPs) in the putative regulatory region of the genes encoding NADPH oxidase 4 catalytic subunit (NOX4) and its regulatory subunit p22phox (CYBA) and their relation with metabolic and histological variables in patients with HCV. One hundred seventy eight naïve HCV patients (49.3% male; 65% HCV genotype 1) with positive HCV RNA were genotyped using specific primers and fluorescent-labeled probes for SNPs rs3017887 in NOX4 and -675 T → A in CYBA. No association was found between the genotype frequencies of NOX4 and CYBA SNPs and inflammation scores or fibrosis stages in the overall population. The presence of the CA + AA genotypes of the NOX4 SNP was nominally associated with a lower alanine aminotransferase (ALT) concentration in the male population (CA + AA = 72.23 ± 6.34 U/L versus CC = 100.22 ± 9.85; mean ± SEM; P = 0.05). The TT genotype of the CYBA SNP was also nominally associated with a lower ALT concentration in the male population (TT = 84.01 ± 6.77 U/L versus TA + AA = 109.67 ± 18.37 U/L; mean ± SEM; P = 0.047). The minor A-allele of the NOX4 SNP was inversely associated with the frequency of metabolic syndrome (MS) in the male population (odds ratio (OR): 0.15; 95% confidence interval (CI): 0.03 to 0.79; P = 0.025). The results suggest that the evaluated NOX4 and CYBA SNPs are not direct genetic determinants of fibrosis in HCV patients, but nevertheless NOX4 rs3017887 SNP could indirectly influence fibrosis susceptibility due to its inverse association with MS in male patients.

  2. The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

    International Nuclear Information System (INIS)

    Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

    2009-01-01

    An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

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

    Science.gov (United States)

    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

  4. The NADPH-oxidase AtRbohI plays a positive role in drought-stress response in Arabidopsis thaliana.

    Science.gov (United States)

    He, Huan; Yan, Jingwei; Yu, Xiaoyun; Liang, Yan; Fang, Lin; Scheller, Henrik Vibe; Zhang, Aying

    2017-09-23

    As the major resource of reactive oxygen species (ROS), the NADPH oxidases (Rbohs) have been shown to play important roles in plant cells under normal growth and stress conditions. Although many family members of Rbohs were studied, little is known about the function of RbohI in Arabidopsis thaliana. Here, we report that exogenous ABA application decreases RbohI expression and mannitol significantly increases RbohI expression at transcript level. The RbohI transcripts were strongly detected in dry seeds and roots. The loss-of-function mutant rbohI exhibited sensitivity to ABA and mannitol stress during germination. Furthermore, the lateral root growth of rbohI was severely inhibited after treatment with mannitol stress. Overexpression of RbohI in Arabidopsis significantly improves the drought tolerance. Moreover, more H 2 O 2 accumulated in RbohI overexpressors than in wild type plants in response to mannitol stress. Our conclusion is that AtRbohI functions in drought-stress response in Arabidopsis thaliana. Copyright © 2017. Published by Elsevier Inc.

  5. Lycopene Inhibits Metastasis of Human Liver Adenocarcinoma SK-Hep-1 Cells by Downregulation of NADPH Oxidase 4 Protein Expression.

    Science.gov (United States)

    Jhou, Bo-Yi; Song, Tuzz-Ying; Lee, Inn; Hu, Miao-Lin; Yang, Nae-Cherng

    2017-08-16

    NADPH oxidase 4 (NOX4), with the sole function to produce reactive oxygen species (ROS), can be a molecular target for disrupting cancer metastasis. Several studies have indicated that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the anti-metastatic effect of lycopene (0.1-5 μM) on human liver adenocarcinoma SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression, with the strongest inhibition of 64.3 ± 10.2% (P lycopene. Lycopene also significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth factor β (TGF-β)-induced metastasis. We found that TGF-β (5 ng/mL) significantly increased migration, invasion, and adhesion activity, the intracellular ROS level, matrix metalloproteinase 9 (MMP-9) and MMP-2 activities, the level of NOX4 protein expression, and NOX activity. All these TGF-β-induced effects were antagonized by the incubation of SK-Hep-1 cells with lycopene (2.5 μM). Using transient transfection of siRNA against NOX4, we found that the downregulation of NOX4 could mimic lycopene by inhibiting cell migration and the activities of MMP-9 and MMP-2 during the incubation with or without TGF-β on SK-Hep-1 cells. The results demonstrate that the downregulation of NOX4 plays a crucial role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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 γ-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. Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase.

    Science.gov (United States)

    Gandara, Ana Caroline Paiva; Torres, André; Bahia, Ana Cristina; Oliveira, Pedro L; Schama, Renata

    2017-03-29

    NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny. We have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H 2 O 2 . Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H 2 O 2 production is considerably diminished in A. aegypti cells. Combining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H 2 O 2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many

  8. Antimutagenic activity of oxidase enzymes

    International Nuclear Information System (INIS)

    Agabeili, R.A.

    1986-01-01

    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

  9. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox family of enzymes

    Directory of Open Access Journals (Sweden)

    Lambeth J David

    2007-07-01

    Full Text Available Abstract Background NADPH-oxidases (Nox and the related Dual oxidases (Duox play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS. Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes. Results We assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to

  10. Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue.

    Science.gov (United States)

    Prince, Paula D; Santander, Yanina A; Gerez, Estefania M; Höcht, Christian; Polizio, Ariel H; Mayer, Marcos A; Taira, Carlos A; Fraga, Cesar G; Galleano, Monica; Carranza, Andrea

    2017-08-01

    Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 β-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 β-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum. Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism. Male Sprague-Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 β-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 β-HSD1, and NADPH/NADP + ratio. Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 β-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Involvement of NADPH oxidase isoforms in the production of O2− manipulated by ABA in the senescing leaves of early-senescence-leaf (esl) mutant rice (Oryza sativa)

    Science.gov (United States)

    Wang, Fubiao; Zhao, Qian; Liu, Jianchao; Cheng, Fangmin

    2018-01-01

    In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (esl) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (Oryza sativa). The temporal expression levels of OsNox isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the esl mutant generated more O2- concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O2- generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O2- generation. The esl mutant exhibited significantly higher expression of OsNox2, OsNox5, OsNox6, and OsNox7 in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of OsNox1, OsNox3, and OsFR07 in the flag leaf of the esl mutant were significantly lower than those in the wild-type cultivar. The expression levels of OsNox2, OsNox5, OsNox6, and OsNox7 were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of OsNox2 and OsNox6 were dependent on the duration of ABA treatment. The inducible expression levels of OsNox5 and OsNox7 were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of OsNox1, OsNox3, and OsFR07 in the detached leaf segments. Therefore, OsNox2, OsNox5, OsNox6, and OsNox7 were probably involved in the ABA-induced O2- generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the

  12. The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells

    Energy Technology Data Exchange (ETDEWEB)

    Guidarelli, Andrea; Fiorani, Mara; Carloni, Silvia; Cerioni, Liana; Balduini, Walter; Cantoni, Orazio, E-mail: orazio.cantoni@uniurb.it

    2016-09-15

    We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O{sub 2}{sup .-}) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5 μM, arsenite elicited selective formation of O{sub 2}{sup .-} in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O{sub 2}{sup .-} triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O{sub 2}{sup .-} because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O{sub 2}{sup .-} mediated by NADPH oxidase. Interestingly, extramitochondrial O{sub 2}{sup .-} triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O{sub 2}{sup .-} availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis. - Highlights: • Mitochondrial superoxide mediates arsenite toxicity in respiration-proficient cells. • NADPH-derived superoxide mediates arsenite toxicity in respiration-deficient cells. • Arsenite causes apoptosis

  13. Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.

    Science.gov (United States)

    White, Caroline N; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Figtree, Gemma A; Rasmussen, Helge H

    2010-04-30

    Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

  14. NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.

    Science.gov (United States)

    Juhasz, Agnes; Markel, Susan; Gaur, Shikha; Liu, Han; Lu, Jiamo; Jiang, Guojian; Wu, Xiwei; Antony, Smitha; Wu, Yongzhong; Melillo, Giovanni; Meitzler, Jennifer L; Haines, Diana C; Butcher, Donna; Roy, Krishnendu; Doroshow, James H

    2017-05-12

    Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O 2 ̇̄ , is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G 1 /S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G 1 /S checkpoint was associated with a significant decrease in cyclin D 1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Sinomenine, a natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase

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    Wilson Belinda

    2007-09-01

    Full Text Available Abstract Background The mechanisms involved in the induction and regulation of inflammation resulting in dopaminergic (DA neurotoxicity in Parkinson's disease (PD are complex and incompletely understood. Microglia-mediated inflammation has recently been implicated as a critical mechanism responsible for progressive neurodegeneration. Methods Mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanisms of sinomenine (SN-mediated anti-inflammatory and neuroprotective effects in both the lipopolysaccharide (LPS- and the 1-methyl-4-phenylpyridinium (MPP+-mediated models of PD. Results SN showed equivalent efficacy in protecting against DA neuron death in rat midbrain neuron-glial cultures at both micro- and sub-picomolar concentrations, but no protection was seen at nanomolar concentrations. The neuroprotective effect of SN was attributed to inhibition of microglial activation, since SN significantly decreased tumor necrosis factor-α (TNF-α, prostaglandin E2 (PGE2 and reactive oxygen species (ROS production by microglia. In addition, from the therapeutic point of view, we focused on sub-picomolar concentration of SN for further mechanistic studies. We found that 10-14 M of SN failed to protect DA neurons against MPP+-induced toxicity in the absence of microglia. More importantly, SN failed to show a protective effect in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX, a key enzyme for extracellular superoxide production in immune cells. Furthermore, we demonstrated that SN reduced LPS-induced extracellular ROS production through the inhibition of the PHOX cytosolic subunit p47phoxtranslocation to the cell membrane. Conclusion Our findings strongly suggest that the protective effects of SN are most likely mediated through the inhibition of microglial PHOX activity. These findings suggest a novel therapy to treat inflammation-mediated neurodegenerative diseases.

  16. Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats

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    Serizawa Ken-ichi

    2011-11-01

    Full Text Available Abstract Background Nicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes. Methods Male Sprague-Dawley rats (6 weeks old were intraperitoneally injected with streptozotocin (STZ, 40 mg/kg, once a day for 3 days to induce diabetes. Nicorandil (15 mg/kg/day and tempol (20 mg/kg/day, superoxide dismutase mimetic were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs were treated with high glucose (35.6 mM, 24 h and reactive oxygen species (ROS production with or without L-NAME (300 μM, apocynin (100 μM or nicorandil (100 μM was measured using fluorescent probes. Results Endothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 ± 1.4%; normal, 19.5 ± 1.7%; n = 6-7. There was a 2.4-fold increase in p47phox expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 ± 2.6%; tempol, 13.3 ± 1.4%; n = 6. Nicorandil significantly inhibited the increased expressions of p47phox and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil

  17. Zinc triggers microglial activation.

    Science.gov (United States)

    Kauppinen, Tiina M; Higashi, Youichirou; Suh, Sang Won; Escartin, Carole; Nagasawa, Kazuki; Swanson, Raymond A

    2008-05-28

    Microglia are resident immune cells of the CNS. When stimulated by infection, tissue injury, or other signals, microglia assume an activated, "ameboid" morphology and release matrix metalloproteinases, reactive oxygen species, and other proinflammatory factors. This innate immune response augments host defenses, but it can also contribute to neuronal death. Zinc is released by neurons under several conditions in which microglial activation occurs, and zinc chelators can reduce neuronal death in animal models of cerebral ischemia and neurodegenerative disorders. Here, we show that zinc directly triggers microglial activation. Microglia transfected with a nuclear factor-kappaB (NF-kappaB) reporter gene showed a severalfold increase in NF-kappaB activity in response to 30 microm zinc. Cultured mouse microglia exposed to 15-30 microm zinc increased nitric oxide production, increased F4/80 expression, altered cytokine expression, and assumed the activated morphology. Zinc-induced microglial activation was blocked by inhibiting NADPH oxidase, poly(ADP-ribose) polymerase-1 (PARP-1), or NF-kappaB activation. Zinc injected directly into mouse brain induced microglial activation in wild-type mice, but not in mice genetically lacking PARP-1 or NADPH oxidase activity. Endogenous zinc release, induced by cerebral ischemia-reperfusion, likewise induced a robust microglial reaction, and this reaction was suppressed by the zinc chelator CaEDTA. Together, these results suggest that extracellular zinc triggers microglial activation through the sequential activation of NADPH oxidase, PARP-1, and NF-kappaB. These findings identify a novel trigger for microglial activation and a previously unrecognized mechanism by which zinc may contribute to neurological disorders.

  18. Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

    Science.gov (United States)

    Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

    2015-09-15

    In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

  19. HMGB1-RAGE pathway drives peroxynitrite signaling-induced IBD-like inflammation in murine nonalcoholic fatty liver disease

    Directory of Open Access Journals (Sweden)

    Varun Chandrashekaran

    2017-10-01

    Full Text Available Recent clinical studies found a strong association of colonic inflammation and Inflammatory bowel disease (IBD-like phenotype with NonAlcoholic Fatty liver Disease (NAFLD yet the mechanisms remain unknown. The present study identifies high mobility group box 1 (HMGB1 as a key mediator of intestinal inflammation in NAFLD and outlines a detailed redox signaling mechanism for such a pathway. NAFLD mice showed liver damage and release of elevated HMGB1 in systemic circulation and increased intestinal tyrosine nitration that was dependent on NADPH oxidase. Intestines from NAFLD mice showed higher Toll like receptor 4 (TLR4 activation and proinflammatory cytokine release, an outcome strongly dependent on the existence of NAFLD pathology and NADPH oxidase. Mechanistically intestinal epithelial cells showed the HMGB1 activation of TLR-4 was both NADPH oxidase and peroxynitrite dependent with the latter being formed by the activation of NADPH oxidase. Proinflammatory cytokine production was significantly blocked by the specific peroxynitrite scavenger phenyl boronic acid (FBA, AKT inhibition and NADPH oxidase inhibitor Apocynin suggesting NADPH oxidase-dependent peroxynitrite is a key mediator in TLR-4 activation and cytokine release via an AKT dependent pathway. Studies to ascertain the mechanism of HMGB1-mediated NADPH oxidase activation showed a distinct role of Receptor for advanced glycation end products (RAGE as the use of inhibitors targeted against RAGE or use of deformed HMGB1 protein prevented NADPH oxidase activation, peroxynitrite formation, TLR4 activation and finally cytokine release. Thus, in conclusion the present study identifies a novel role of HMGB1 mediated inflammatory pathway that is RAGE and redox signaling dependent and helps promote ectopic intestinal inflammation in NAFLD.

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

    Science.gov (United States)

    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-1β, 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-1β 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-κB, 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 μM), and IL-1β 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-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB 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.

  1. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

    Science.gov (United States)

    González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

    2014-01-01

    In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

  2. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees

    Directory of Open Access Journals (Sweden)

    Alberto eGonzález

    2014-10-01

    Full Text Available In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control, with OC kappa at 1 mg mL-1, or treated with inhibitors of NAD(PH, ascorbate (ASC and glutathione (GSH syntheses and thioredoxin reductase (TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS, adenosine 5´-phosphosulfate reductase (APR, involved in C, N and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism and growth in Eucalyptus trees.

  3. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 exposed renal ischemia/reperfusion injury in rats

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    Zhiming Li

    2015-01-01

    Full Text Available This study was designed to evaluate whether NADPH oxidase inhibitor (apocynin preconditioning induces expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 to protect against renal ischemia/reperfusion (I/R injury (RI/RI in rats. Rats were pretreated with 50 mg/kg apocynin, then subjected to 45 min ischemia and 24 h reperfusion. The results indicated that apocynin preconditioning improved the recovery of renal function and nitroso-redox balance, reduced oxidative stress injury and inflammation damage, and upregulated expression of SHP-1 as compared to RI/RI group. Therefore our study demonstrated that apocynin preconditioning provided a protection to the kidney against I/R injury in rats partially through inducing expression of SHP-1.

  4. Ginsenoside Re protects against phencyclidine-induced behavioral changes and mitochondrial dysfunction via interactive modulation of glutathione peroxidase-1 and NADPH oxidase in the dorsolateral cortex of mice.

    Science.gov (United States)

    Tran, The-Vinh; Shin, Eun-Joo; Dang, Duy-Khanh; Ko, Sung Kwon; Jeong, Ji Hoon; Nah, Seung-Yeol; Jang, Choon-Gon; Lee, Yu Jeung; Toriumi, Kazuya; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2017-12-01

    We investigated whether ginsenoside Re (Re) modulates phencyclidine (PCP)-induced sociability deficits and recognition memory impairments to extend our recent finding. We examined the role of GPx-1 gene in the pharmacological activity of Re against mitochondrial dysfunction induced by PCP in the dorsolateral cortex of mice. Since mitochondrial oxidative stress activates NADPH oxidase (PHOX), we applied PHOX inhibitor apocynin for evaluating interactive modulation between GPx-1 and PHOX against PCP neurotoxicity. Sociability deficits and recognition memory impairments induced by PCP were more pronounced in GPx-1 knockout (KO) than in wild type (WT) mice. PCP-induced mitochondrial oxidative stress, mitochondrial dysfunction, and membrane translocation of p47phox were more evident in GPx-1 KO than in WT. Re treatment significantly attenuated PCP-induced neurotoxic changes. Re also significantly attenuated PCP-induced sociability deficits and recognition memory impairments. The attenuation by Re was comparable to that by apocynin. The attenuation was more obvious in GPx-1 KO than in WT. Importantly, apocynin did not show any additional positive effects on the neuroprotective activity of Re, indicating that PHOX is a molecular target for therapeutic activity of Re. Our results suggest that Re requires interactive modulation between GPx activity and PHOX (p47phox) to exhibit neuroprotective potentials against PCP insult. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Seasonal superoxide overproduction and endothelial activation in guinea-pig heart; seasonal oxidative stress in rats and humans.

    Science.gov (United States)

    Konior, Anna; Klemenska, Emilia; Brudek, Magdalena; Podolecka, Ewa; Czarnowska, Elżbieta; Beręsewicz, Andrzej

    2011-04-01

    Seasonality in endothelial dysfunction and oxidative stress was noted in humans and rats, suggesting it is a common phenomenon of a potential clinical relevance. We aimed at studying (i) seasonal variations in cardiac superoxide (O(2)(-)) production in rodents and in 8-isoprostane urinary excretion in humans, (ii) the mechanism of cardiac O(2)(-) overproduction occurring in late spring/summer months in rodents, (iii) whether this seasonal O(2)(-)-overproduction is associated with a pro-inflammatory endothelial activation, and (iv) how the summer-associated changes compare to those caused by diabetes, a classical cardiovascular risk factor. Langendorff-perfused guinea-pig and rat hearts generated ~100% more O(2)(-), and human subjects excreted 65% more 8-isoprostane in the summer vs. other seasons. Inhibitors of NADPH oxidase, xanthine oxidase, and NO synthase inhibited the seasonal O(2)(-)-overproduction. In the summer vs. other seasons, cardiac NADPH oxidase and xanthine oxidase activity, and protein expression were increased, the endothelial NO synthase and superoxide dismutases were downregulated, and, in guinea-pig hearts, adhesion molecules upregulation and the endothelial glycocalyx destruction associated these changes. In guinea-pig hearts, the summer and a streptozotocin-induced diabetes mediated similar changes, yet, more severe endothelial activation associated the diabetes. These findings suggest that the seasonal oxidative stress is a common phenomenon, associated, at least in guinea-pigs, with the endothelial activation. Nonetheless, its biological meaning (regulatory vs. deleterious) remains unclear. Upregulated NADPH oxidase and xanthine oxidase and uncoupled NO synthase are the sources of the seasonal O(2)(-)-overproduction. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. NADPH oxidase 1 deficiency alters caveolin phosphorylation and angiotensin II-receptor localization in vascular smooth muscle.

    Science.gov (United States)

    Basset, Olivier; Deffert, Christine; Foti, Michelangelo; Bedard, Karen; Jaquet, Vincent; Ogier-Denis, Eric; Krause, Karl-Heinz

    2009-10-01

    The superoxide-generating NADPH oxidase NOX1 is thought to be involved in signaling by the angiotensin II-receptor AT1R. However, underlying signaling steps are poorly understood. In this study, we investigated the effect of AngII on aortic smooth muscle from wild-type and NOX1-deficient mice. NOX1-deficient cells showed decreased basal ROS generation and did not produce ROS in response to AngII. Unexpectedly, AngII-dependent Ca(2+) signaling was markedly decreased in NOX1-deficient cells. Immunostaining demonstrated that AT1R was localized on the plasma membrane in wild-type, but intracellularly in NOX1-deficient cells. Immunohistochemistry and immunoblotting showed a decreased expression of AT1R in the aorta of NOX1-deficient mice. To investigate the basis of the abnormal AT1R targeting, we studied caveolin expression and phosphorylation. The amounts of total caveolin and of caveolae were not different in NOX1-deficient mice, but a marked decrease occurred in the phosphorylated form of caveolin. Exogenous H(2)O(2) or transfection of a NOX1 plasmid restored AngII responses in NOX1-deficient cells. Based on these findings, we propose that NOX1-derived reactive oxygen species regulate cell-surface expression of AT1R through mechanisms including caveolin phosphorylation. The lack cell-surface AT1R expression in smooth muscle could be involved in the decreased blood pressure in NOX1-deficient mice.

  7. Variations in creatine kinase activity and reactive oxygen species levels are involved in capacitation of bovine spermatozoa.

    Science.gov (United States)

    Córdoba, M; Pintos, L; Beconi, M T

    2008-12-01

    The generation of reactive oxygen species (ROS) is associated with some factors such as oxidative substrate sources, mitochondrial function and NAD(P)H oxidase activity. In bovine spermatozoa, heparin capacitation produces a respiratory burst sensitive to diphenyleneiodonium (DPI). Creatine kinase (CK) is related to extramitochondrial ATP disponibility. Our purpose was to determine the variation in ROS level and its relation with NAD(P)H oxidase sensitive to DPI and CK participation, as factors involved in redox state and energy generation in capacitation. The chlortetracycline technique was used to evaluate capacitation. CK activity and ROS level were measured by spectrophotometry and spectrofluorometry respectively. The capacitation percentage was increased by heparin or quercetin treatment (P level as control (238.62 +/- 23.47 arbitrary units per 10(8) spermatozoa) (P > 0.05). CK activity decreased by 50% with heparin or quercetin (P level variations were observed in heparin- or quercetin-treated samples (P bovine spermatozoa, capacitation requires equilibrium between oxidative damage susceptibility and ROS levels. CK activity is associated with redox state variation and energy sources. In conclusion, capacitation induction depends on NADPH oxidase and the shuttle creatine-creatine phosphate, both sensitive to DPI.

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

    Energy Technology Data Exchange (ETDEWEB)

    Blacker, Thomas S., E-mail: t.blacker@ucl.ac.uk [Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London, London WC1E 6BT (United Kingdom); Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom); Research Department of Cell and Developmental Biology, University College London, London WC1E 6BT (United Kingdom); Marsh, Richard J., E-mail: richard.marsh@ucl.ac.uk [Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom); Duchen, Michael R., E-mail: m.duchen@ucl.ac.uk [Research Department of Cell and Developmental Biology, University College London, London WC1E 6BT (United Kingdom); Bain, Angus J., E-mail: a.bain@ucl.ac.uk [Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London, London WC1E 6BT (United Kingdom); Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom)

    2013-08-30

    Highlights: ► NADH and NADPH have a high rate of non-radiative excited state decay. ► Conformational relaxation is shown to be a significant non-radiative pathway. ► The Kramers equation describes the barrier crossing dynamics of the relaxation. ► Conformational restriction upon enzyme binding will alter NAD(P)H lifetimes. - Abstract: 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.

  9. Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

    Science.gov (United States)

    de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

    2017-11-01

    Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

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

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    Xiaohui eLi

    2015-06-01

    Full Text Available NADPH oxidases (also known as respiratory burst oxidase homologues, Rbohs are the 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 (VIGS-based silencing of SlRbohB resulted in reduced resistance to B. cinerea but silencing of each of other SlRbohs did not affect the resistance. The SlRbohB-silenced plants accumulated more ROS and attenuated expression of defense genes after infection of B. cinerea than the nonsilenced plants. 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 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.

  11. Burkholderia pseudomallei Evades Nramp1 (Slc11a1- and NADPH Oxidase-Mediated Killing in Macrophages and Exhibits Nramp1-Dependent Virulence Gene Expression

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    Veerachat Muangsombut

    2017-08-01

    Full Text Available Bacterial survival in macrophages can be affected by the natural resistance-associated macrophage protein 1 (Nramp1; also known as solute carrier family 11 member a1 or Slc11a1 which localizes to phagosome membranes and transports divalent cations, including iron. Little is known about the role of Nramp1 in Burkholderia infection, in particular whether this differs for pathogenic species like Burkholderia pseudomallei causing melioidosis or non-pathogenic species like Burkholderia thailandensis. Here we show that transfected macrophages stably expressing wild-type Nramp1 (Nramp1+ control the net replication of B. thailandensis, but not B. pseudomallei. Control of B. thailandensis was associated with increased cytokine responses, and could be abrogated by blocking NADPH oxidase-mediated production of reactive oxygen species but not by blocking generation of reactive nitrogen species. The inability of Nramp1+ macrophages to control B. pseudomallei was associated with rapid escape of bacteria from phagosomes, as indicated by decreased co-localization with LAMP1 compared to B. thailandensis. A B. pseudomallei bipB mutant impaired in escape from phagosomes was controlled to a greater extent than the parent strain in Nramp1+ macrophages, but was also attenuated in Nramp1− cells. Consistent with reduced escape from phagosomes, B. thailandensis formed fewer multinucleated giant cells in Nramp1+ macrophages at later time points compared to B. pseudomallei. B. pseudomallei exhibited elevated transcription of virulence-associated genes of Type VI Secretion System cluster 1 (T6SS-1, the Bsa Type III Secretion System (T3SS-3 and the bimA gene required for actin-based motility in Nramp1+ macrophages. Nramp1+ macrophages were found to contain decreased iron levels that may impact on expression of such genes. Our data show that B. pseudomallei is able to evade Nramp1- and NADPH oxidase-mediated killing in macrophages and that expression of virulence

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

    Energy Technology Data Exchange (ETDEWEB)

    Magnani, Natalia D.; Marchini, Timoteo; Vanasco, Virginia [Instituto de Bioquímica Medicina Molecular (IBIMOL-UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (Argentina); Tasat, Deborah R. [CESyMA, Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); Alvarez, Silvia [Instituto de Bioquímica Medicina Molecular (IBIMOL-UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (Argentina); Evelson, Pablo, E-mail: pevelson@ffyb.uba.ar [Instituto de Bioquímica Medicina Molecular (IBIMOL-UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (Argentina)

    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

  13. Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

    Science.gov (United States)

    Nagasawa, Kazuki; Kakuda, Taichi; Higashi, Youichirou; Fujimoto, Sadaki

    2007-12-18

    The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

  14. NADPH-d activity in rat thymus after the application of retinoid acid

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    F. Dorko

    2012-02-01

    Full Text Available The aim of this work was to determine the localization of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d activity as the marker for synthesis of nitric oxide synthase (NOS in the rat thymus after the application of retinoid acid (RA on 1st, 7th, 14th and 21st days of gestation. The given results can build the basis for understanding of the role of NOS in rat thymus. NADPH-d positive cells were represented with dark-blue color and were localized on corticomedullar junction of the thymus. These cells were of different intensity of coloring and were shaped in oval, circle or irregular forms. NADPH-d positive nerve fibers were observed in perivascular topography. They were marked more strongly in the case of control group. The result of application of RA to gravid rats was that the birth weights of newborn rats and their thymuses were smaller, but without statistically significance.

  15. Studying neuroprotective effect of Atorvastatin as a small molecule drug on high glucose-induced neurotoxicity in undifferentiated PC12 cells: role of NADPH oxidase.

    Science.gov (United States)

    Rayegan, Samira; Dehpour, Ahmad Reza; Sharifi, Ali Mohammad

    2017-02-01

    Overproduction of reactive oxygen species (ROS) by NADPH oxidase (NOX) activation has been considered the essential mechanism induced by hyperglycemia in various tissues. However, there is no comprehensive study on the role of NOXs in high glucose (HG)-induced toxic effect in neural tissues. Recently, a therapeutic strategy in oxidative related pathologies has been introduced by blocking the undesirable actions of NOX enzymes by small molecules. The protective roles of Statins in ameliorating oxidative stress by NOX inhibition have been shown in some tissues except neural. We hypothesized then, that different NOXs may have role in HG-induced neural cell injury. Furthermore, we postulate that Atorvastatin as a small molecule may modulate this NOXs activity to protect neural cells. Undifferentiated PC12 cells were treated with HG (140 mM/24 h) in the presence and absence of Atorvastatin (1 μM/96 h). The cell viability was measured by MTT assay and the gene and protein expressions profile of NOX (1-4) were determined by RT-PCR and western blotting, respectively. Levels of ROS and malondialdehyde (MDA) were also evaluated. Gene and protein expression levels of NOX (1-4) and consequently ROS and MDA levels were elevated in HG-treated PC12 cells. Atorvastatin could significantly decrease HG-induced NOXs, ROS and MDA elevation and improve impaired cell viability. It can be concluded that HG could elevate NOXs activity, ROS and MDA levels in neural tissues and Atorvastatin as a small molecule NOX inhibitor drug may prevent and delay diabetic complications, particularly neuropathy.

  16. Platelet monoamine oxidase: specific activity and turnover number in headache

    International Nuclear Information System (INIS)

    Summers, K.M.; Brown, G.K.; Craig, I.W.; Peatfield, R.; Rose, F.C.

    1982-01-01

    Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [ 3 H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction. (Auth.)

  17. Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity

    International Nuclear Information System (INIS)

    Cui, Changtai T.; Uriu-Adams, Janet Y.; Tchaparian, Eskouhie H.; Keen, Carl L.; Rucker, Robert B.

    2004-01-01

    Selected indices of copper metabolism in weanling rats and fibroblast cultures were progressively altered in response to increased levels of sodium metavanadate. In diets, vanadium was added in amounts ranging from 0 to 80 μg V/g of diet, that is, 0-1.6 μmol V/g of diet. In fibroblast cultures, vanadium ranged from 0 to 400 nmol V/ml. The inhibition of P-ATPase-7A activity by metavanadate, important to copper egress from cells, was a primary focus. In skin, and tendon, the copper concentration was increased in response to increased dietary levels of metavanadate, whereas lysyl oxidase activity, a secreted cuproprotein, was reduced. The reduction in lysyl oxidase activity was also accompanied by reduced redox cycling potential of isolated fractions of lysyl oxidase, presumably due to reduced lysyltyrosyl quinone (LTQ) formation at the active site of lysyl oxidase. In contrast, liver copper concentrations and plasma ceruloplasmin activity were not affected by metavanadate exposure. However, semicarbazide-sensitive benzylamine oxidase (SCBO) activity, which was taken as an indirect measure of vascular adhesive protein-1 (VAP-1), was increased. In cultured fibroblasts, cellular copper was also increased and lysyl oxidase decreased in response to metavanadate. Moreover, the steady-state levels of atp7a and lysyl oxidase mRNAs were not affected by addition of metavanadate to culture medium up to 200 nmol/ml. Taken together, these data suggest that pathways involving copper egress and lysyl oxidase activation are particularly sensitive to metavanadate exposure through processes that are predominately posttranslational

  18. Defects in Nicotinamide-adenine Dinucleotide Phosphate Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel DiseaseSummary

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    Patti Hayes

    2015-09-01

    Full Text Available Background & Aims: Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD. Reactive oxygen species (ROS generated by nicotinamide-adenine dinucleotide phosphate (NADPH oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD. Methods: After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 59 children with very early onset inflammatory bowel disease (VEOIBD, the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli. Results: We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. Patients with both NOX1 and DUOX2 variants showed abnormal Paneth cell metaplasia. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni. Conclusions: This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD. Keywords: Inflammatory Bowel Disease, NADPH Oxidase

  19. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-11-02

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  20. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  1. Hydrogen sulfide inhibits high glucose-induced NADPH oxidase 4 expression and matrix increase by recruiting inducible nitric oxide synthase in kidney proximal tubular epithelial cells.

    Science.gov (United States)

    Lee, Hak Joo; Lee, Doug Yoon; Mariappan, Meenalakshmi M; Feliers, Denis; Ghosh-Choudhury, Goutam; Abboud, Hanna E; Gorin, Yves; Kasinath, Balakuntalam S

    2017-04-07

    High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H 2 S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H 2 S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H 2 S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H 2 S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N (ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H 2 S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H 2 S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

    Science.gov (United States)

    Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

    2011-08-01

    Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

  3. Structure and activity of Aspergillus nidulans copper amine oxidase

    DEFF Research Database (Denmark)

    McGrath, Aaron P; Mithieux, Suzanne M; Collyer, Charles A

    2011-01-01

    Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related...... enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer...... with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity....

  4. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

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    Ajantha Nadesalingam

    2018-03-01

    Full Text Available Tonicity of saline (NaCl is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA, Gram-negative bacterial cell wall component lipopolysaccharide (LPS, calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus, and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus. We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12, and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol. This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  5. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

    Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  6. Nox1 oxidase suppresses influenza a virus-induced lung inflammation and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Stavros Selemidis

    Full Text Available Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1(-/y mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×10(4 PFU influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1(-/y mice resulted in significantly greater: loss of bodyweight (Day 3; BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1(-/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1(-/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8(+ and CD4(+ T lymphocytes, and of Tregs were similar between WT and Nox1(-/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear

  7. Cluster Differentiating 36 (CD36) Deficiency Attenuates Obesity-Associated Oxidative Stress in the Heart.

    Science.gov (United States)

    Gharib, Mohamed; Tao, Huan; Fungwe, Thomas V; Hajri, Tahar

    2016-01-01

    Obesity is often associated with a state of oxidative stress and increased lipid deposition in the heart. More importantly, obesity increases lipid influx into the heart and induces excessive production of reactive oxygen species (ROS) leading to cell toxicity and metabolic dysfunction. Cluster differentiating 36 (CD36) protein is highly expressed in the heart and regulates lipid utilization but its role in obesity-associated oxidative stress is still not clear. The aim of this study was to determine the impact of CD36 deficiency on cardiac steatosis, oxidative stress and lipotoxicity associated with obesity. Studies were conducted in control (Lean), obese leptin-deficient (Lepob/ob) and leptin-CD36 double null (Lepob/obCD36-/-) mice. Compared to lean mice, cardiac steatosis, and fatty acid (FA) uptake and oxidation were increased in Lepob/ob mice, while glucose uptake and oxidation was reduced. Moreover, insulin resistance, oxidative stress markers and NADPH oxidase-dependent ROS production were markedly enhanced. This was associated with the induction of NADPH oxidase expression, and increased membrane-associated p47phox, p67phox and protein kinase C. Silencing CD36 in Lepob/ob mice prevented cardiac steatosis, increased insulin sensitivity and glucose utilization, but reduced FA uptake and oxidation. Moreover, CD36 deficiency reduced NADPH oxidase activity and decreased NADPH oxidase-dependent ROS production. In isolated cardiomyocytes, CD36 deficiency reduced palmitate-induced ROS production and normalized NADPH oxidase activity. CD36 deficiency prevented obesity-associated cardiac steatosis and insulin resistance, and reduced NADPH oxidase-dependent ROS production. The study demonstrates that CD36 regulates NADPH oxidase activity and mediates FA-induced oxidative stress.

  8. Expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and mitochondrial oxidative phosphorylation in septic patients.

    Directory of Open Access Journals (Sweden)

    Laura A Nucci

    Full Text Available Sepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients.Blood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0, and after 7 days (D7, N = 10 of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant.Twenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients.Our data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by changes in JAK-STAT modulation, had prominent changes in

  9. Microvascular Endothelial Dysfunction in Sedentary, Obese Humans is mediated by NADPH Oxidase; Influence of Exercise Training

    Science.gov (United States)

    La Favor, Justin D.; Dubis, Gabriel S.; Yan, Huimin; White, Joseph D.; Nelson, Margaret A.M.; Anderson, Ethan J.; Hickner, Robert C.

    2016-01-01

    Objective The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and to determine the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. Approach and Results Young, sedentary men and women were divided into lean (BMI 18–25; n=14), intermediate (BMI 28–32.5; n=13), and obese (BMI 33–40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared to both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase (Nox) inhibitor, lowered (normalized) H2O2 and superoxide levels and reversed microvascular endothelial dysfunction in obese subjects. Following 8-weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the Nox subunits p22phox, p47phox, and p67phox were increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. Conclusions This study implicates Nox as a source of excessive ROS production in skeletal muscle of obese individuals, and links excessive Nox derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies. PMID:27765769

  10. Plasma diamine oxidase activity in asthmatic children

    Directory of Open Access Journals (Sweden)

    Kyoichiro Toyoshima

    1996-01-01

    Full Text Available Histamine plays an important role in the development of asthmatic symptoms. Diamine oxidase (DAO histaminase, which inactivates histamine, is located in the intestine and kidney and is released into plasma. Plasma DAO activity in asthmatic children was measured by a recently developed high performance liquid chromatographic method using histamine as the DAO substrate. Diamine oxidase activity was higher in severely asthmatic children than in those with mild asthma. A time course study during the acute exacerbation phase revealed that DAO activity rose during acute asthmatic attacks and then decreased gradually over several days. Although the mechanisms of plasma DAO activity increase during acute asthmatic attacks could not be explained, data showed that plasma DAO activity is an important index of histamine metabolism in asthmatics and may relate to some mechanisms of acute exacerbation of airway inflammation. Consequently, fluctuations in plasma DAO can be used as one of various indices of instability in management of asthma.

  11. Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

    Science.gov (United States)

    Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

    2015-01-20

    Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

  12. Serum diamine oxidase activity in patients with histamine intolerance.

    Science.gov (United States)

    Manzotti, G; Breda, D; Di Gioacchino, M; Burastero, S E

    2016-03-01

    Intolerance to various foods, excluding bona fide coeliac disease and lactose intolerance, represents a growing cause of patient visits to allergy clinics.Histamine intolerance is a long-known, multifaceted clinical condition triggered by histamine-rich foods and alcohol and/or by drugs that liberate histamine or block diamine oxidase (DAO), the main enzyme involved in the metabolism of ingested histamine. Histamine limitation diets impose complex, non-standardized restrictions that may severely impact the quality of life of patients. We retrospectively evaluated 14 patients who visited allergy outpatient facilities in northern Italy with a negative diagnosis for IgE-mediated food hypersensitivity, coeliac disease, conditions related to gastric hypersecretion, and systemic nickel hypersensitivity, and who previously underwent a histamine limitation diet with benefits for their main symptoms. Serum diamine oxidase levels and the clinical response to diamine oxidase supplementation were investigated. We found that 10 out of 14 patients had serum DAO activityintolerance. Moreover, 13 out of 14 patients subjectively reported a benefit in at least one of the disturbances related to food intolerances following diamine oxidase supplementation. The mean value (±SD) of diamine oxidase activity in the cohort of patients with histamine intolerance symptoms was 7.04±6.90 U/mL compared to 39.50±18.16 U/mL in 34 healthy controls (P=0.0031). In patients with symptoms triggered by histamine-rich food, measuring the serum diamine oxidase activity can help identify subjects who can benefit from a histamine limitation diet and/or diamine oxidase supplementation.Properly designed, controlled studies investigating histamine intolerance that include histamine provocation are indispensable for providing insights into the area of food intolerances, which are currently primarily managed with non-scientific approaches in Italy. © The Author(s) 2015.

  13. The HIV-1 Nef protein and phagocyte NADPH oxidase activation

    DEFF Research Database (Denmark)

    Vilhardt, Frederik; Plastre, Olivier; Sawada, Makoto

    2002-01-01

    of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation...

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

    Science.gov (United States)

    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.

  15. Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

    Science.gov (United States)

    Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

    2017-10-01

    In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. β-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger.

    Science.gov (United States)

    Vitiello, Damien; Boissière, Julien; Doucende, Grégory; Gayrard, Sandrine; Polge, Anne; Faure, Patrice; Goux, Aurélie; Tanguy, Stéphane; Obert, Philippe; Reboul, Cyril; Nottin, Stéphane

    2011-11-01

    Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that β-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the β-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P stress from the Nox enzyme.

  17. Histochemical characterization, distribution and morphometric analysis of NADPH diaphorase neurons in the spinal cord of the agouti

    Directory of Open Access Journals (Sweden)

    Marco Aurelio M Freire

    2008-05-01

    Full Text Available We evaluated the neuropil distribution of the enzymes NADPH diaphorase (NADPH-d and cytochrome oxidase (CO in the spinal cord of the agouti, a medium-sized diurnal rodent, together with the distribution pattern and morphometrical characteristics of NADPH-d reactive neurons across different spinal segments. Neuropil labeling pattern was remarkably similar for both enzymes in coronal sections: reactivity was higher in regions involved with pain processing. We found two distinct types of NADPH-d reactive neurons in the agouti’s spinal cord: type I neurons had large, heavily stained cell bodies while type II neurons displayed relatively small and poorly stained somata. We concentrated our analysis on type I neurons. These were found mainly in the dorsal horn and around the central canal of every spinal segment, with a few scattered neurons located in the ventral horn of both cervical and lumbar regions. Overall, type I neurons were more numerous in the cervical region. Type I neurons were also found in the white matter, particularly in the ventral funiculum. Morphometrical analysis revealed that type I neurons located in the cervical region have dendritic trees that are more complex than those located in both lumbar and thoracic regions. In addition, NADPH-d cells located in the ventral horn had a larger cell body, especially in lumbar segments. The resulting pattern of cell body and neuropil distribution is in accordance with proposed schemes of segregation of function in the mammalian spinal cord.

  18. Effects of telmisartan on the expression of NADPH oxidase subunits in the myocardium of type 2 diabetic rats

    Directory of Open Access Journals (Sweden)

    Jia-wei LI

    2011-10-01

    Full Text Available Objective To explore the effect of telmisartan on the expression of NADPH oxidase subunits p22phox and NOX4 in the myocardiam of type 2 diabetic rats.Methods Thirty-six male Wistar rats were randomly divided into two groups: normal control group(group A,n=10,diabetic model group(n=26.Type 2 diabetic model was established by high-fat and high-sugar diet followed by intraperitoneal injection of a low dose of streptozotocin(STZ.After the model was reproduced successfully,20 diabetic rats were randomly divided into diabetic subgroup(group B,n=10 and telmisartan-treated subgroup(group C,n=10.Rats in group C were orally administered telmisartan(5mg/kg/d,and rats in group A and B were given equivalent volume of normal saline.All rats were sacrificed 12 weeks after treatment.The mRNA expressions of myocardial p22phox and NOX4 were detected by real-time fluorescence quantitative PCR,and the protein expressions of myocardial connective tissue growth factor(CTGF and copper-zinc-superoxide dismutase(Cu-Zn-SOD were detected by immunohistochemical staining.Results Compared with group A,the ratio of heart/body weight,the mRNA expression of myocardial p22phox and NOX4,and the protein expression of myocardial CTGF increased significantly in group B,and the protein expression of myocardial Cu-Zn-SOD decreased significantly(P 0.05.Conclusions Telmisartan can down-regulate the over-expression of myocardial NOX4 and p22phox mRNA in type 2 diabetic rats,lessen the myocardial damage induced by oxidative stress,thus plays a protective role in the myocardium of diabetic rats.

  19. Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase.

    Science.gov (United States)

    Nestler, Josefine; Liu, Sanzhen; Wen, Tsui-Jung; Paschold, Anja; Marcon, Caroline; Tang, Ho Man; Li, Delin; Li, Li; Meeley, Robert B; Sakai, Hajime; Bruce, Wesley; Schnable, Patrick S; Hochholdinger, Frank

    2014-09-01

    Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups 'response to oxidative stress' and 'cellulose biosynthesis' were most prominently represented. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  20. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

    Science.gov (United States)

    Gaurav, Rohit; Bewtra, Againdra K; Agrawal, Devendra K

    2015-08-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

  1. Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition.

    Science.gov (United States)

    Chia, Karin K M; Liu, Chia-Chi; Hamilton, Elisha J; Garcia, Alvaro; Fry, Natasha A; Hannam, William; Figtree, Gemma A; Rasmussen, Helge H

    2015-08-15

    Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O2 (·-)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease β1-Na(+)-K(+) pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. Copyright © 2015 the American Physiological Society.

  2. Innervation of the sheep pineal gland by nonsympathetic nerve fibers containing NADPH-diaphorase activity

    DEFF Research Database (Denmark)

    López-Figueroa, Manuel O.; Ravault, Jean-Paul; Cozzi, Bruno

    1997-01-01

    Neuroanatomy, NADPH-diaphorase, nitric oxide, innervation, superior cervical ganglionectomy, neuropeptide Y.......Neuroanatomy, NADPH-diaphorase, nitric oxide, innervation, superior cervical ganglionectomy, neuropeptide Y....

  3. Differential levels of metabolic activity in isolated versus confluent/partially confluent HeLa cells are analyzed by autofluorescent NAD(P)H using multi-photon FLIM microscopy

    Science.gov (United States)

    Chandler, Andrea; Chandler, Aaron; Wallrabe, Horst; Periasamy, Ammasi

    2017-02-01

    NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ1), the bound version (τ2) a longer lifetime. FLIM's unique capability to establish inter alia the relative fractions of τ1 (a1%) and τ2 (a2%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

  4. Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

    Science.gov (United States)

    Moosavi-Nejad, S Z; Rezaei-Tavirani, M; Padiglia, A; Floris, G; Moosavi-Movahedi, A A

    2001-07-01

    Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

  5. Alzheimer's disease: a gas model. The NADPH oxidase-Nitric Oxide system as an antibubble biomachinery.

    Science.gov (United States)

    Denis, Pierre A

    2013-12-01

    Alzheimer's disease (AD) is a neurodegenerative disease of unknown origin. The pathological lesions that define AD would be linked to the insidious accumulation of nitrogen, having invaded the brain interstitial fluid (ISF) from the blood via the physiological cycling pool of vascular glucose transporters (GLUT-1). According to this hypothesis, the nitrogen nanobubbles, being chemically inert and actually indestructible for human beings, can not escape from the ISF anymore. They would exert a huge and deleterious pressure against cellular components, especially in microglia and in astrocytes. They could enhance the existing cell oxygen anisotropy, which might enhance the natural bubble nucleation of O2-2O2 in cells or in mitochondria. Indeed, with the help of a new symbolic representation for gas nuclei in chemical reactions, the NADPH oxidase-NO system is identified for the first time, as an antibubble biomachinery, able to break O2-2O2 bubbles up as it releases superoxide O2-. Superoxide is considered as a quantum bubble, which collapses through the reactivity of the gaseous NO radical. Their combination in soluble peroxinitrite provides the change from one state of matter to another, avoiding any risk of a bubble enlargement, and finally avoiding the risk of enzyme crowding or of a bulk pressure variation. However, a bubble is expected to entrap Nitric Oxide (NO), which leads theoretically to a decrease in its bioavailability, and is expected to trigger a guanylyl-cyclase-mediated inflammatory cascade, that could explain the inflammation in AD. In vitro, any increase in the hydrostatic pressure has already been linked to the microtubule disorganization. The amyloid deposits, also known as senile plaques, would behave as a sponge toward ISF nitrogen; Aβ is considered as a foam-stabilizing agent. By taking the shape of cerebral amyloid angiopathy, the amyloid could confine the nitrogen leak from the blood, and progressively insulate the Blood-Brain Barrier

  6. Study of Possible Mechanisms Involved in the Inhibitory Effects of Coumarin Derivatives on Neutrophil Activity

    Czech Academy of Sciences Publication Activity Database

    Drábiková, K.; Perečko, T.; Nosáľ, R.; Harmatha, Juraj; Šmidrkal, J.; Jančinová, V.

    2013-01-01

    Roč. 2013, October (2013), 136570/1-136570/10 ISSN 1942-0900 Institutional support: RVO:61388963 Keywords : protein-kinase-C * phagocyte NADPH oxidase * antioxidant activity Subject RIV: CC - Organic Chemistry Impact factor: 3.363, year: 2013 http://www.hindawi.com/journals/omcl/2013/136570/

  7. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    International Nuclear Information System (INIS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-01-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na 2 SeO 3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K m ) values and maximal reaction velocity (V max ) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  8. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    Science.gov (United States)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-03-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  9. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    Williams, C. David; Bajt, Mary Lynn [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Sharpe, Matthew R. [Department of Internal Medicine, University of Kansas Hospital, Kansas City, KS (United States); McGill, Mitchell R. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  10. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome

    DEFF Research Database (Denmark)

    Galley, H F; Davies, Michael Jonathan; Webster, N R

    1996-01-01

    OBJECTIVE: To determine xanthine oxidase activity, free radical concentrations, and lipid peroxidation in patients with sepsis syndrome compared with noninfected critically ill patients. DESIGN: A prospective observational study. SETTING: A nine-bed intensive care unit in a university teaching......). CONCLUSIONS: Patients with sepsis have xanthine oxidase activation, high free-radical concentrations, and evidence of free radical damage. The finding that xanthine oxidase activity was lower in those patients who died, coupled with increased lactate concentrations implies more severe ischemia with incomplete...... to the Acute Physiology and Chronic Health Evaluation (APACHE) II score or to the presence of organ dysfunction. The mean ascorbyl radical concentration (arbitrary units) determined by electron paramagnetic resonance following spin trapping was increased in patients compared with healthy subjects (p

  11. Radio-isotopic determination of platelet monoamine oxidase and regulation of its activity by an indigenous drug

    International Nuclear Information System (INIS)

    Dubey, G.P.; Srivastava, V.K.; Agrawal, A.; Udupa, K.N.

    1988-01-01

    Platelet monoamine oxidase is a mitochondrial enzyme taking part in the deamination reaction of total catecholamine. Recent studies of monoamine oxidase inhibitors have gained its importance in the control of variety of psychosomatic disorders like mental depression, arterial hypertension and anxiety neurosis. 30 apparently normal individuals and 42 diagnosed cases of essential hypertension were selected for the present study. The platelet monoamine oxidase activity was measured by using 14 C-tryptamine bisuccinate. Comparatively low activity of platelet monoamine oxidase was noticed in hypertension cases than in the normal. After oral administration of an indigenous drug 'Geriforte' for three months, a significant rise in platelet monoamine oxidase activity was noticed in hypertension cases. It can be concluded that this indigenous formulation has the capacity to regulate the monoamine oxidase activity, as such, it may provide an alternative remedy in the management of psychosomatic disorders. (author). 11 refs

  12. Antioxidant Activity of Some Plant Extracts Towards Xanthine Oxidase, Lipoxygenase and Tyrosinase

    Directory of Open Access Journals (Sweden)

    Pi-Yu Chen

    2009-08-01

    Full Text Available Natural products have the potential to be developed into new drugs for the treatment of various diseases. The aim of the present study was to screen the antioxidant activities of some common edible fruits, garden plants and medicinal plants indigenous to Taiwan. This was performed by assessing the activities of lipoxygenase, xanthine oxidase and tyrosinase following incubation with extracts from these plants. A further aim was to use HPLC-DAD and tyrosinase to chromatographically identify the antioxidative constituents obtained from an extract exhibiting strong antioxidative properties. The acetone extracts of 27 cultivated plant species from Taiwan were tested for antioxidant activities towards xanthine oxidase, tyrosinase and lipoxygenase using spectrophotometric assays. Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea showed the highest xanthine oxidase inhibitory activities. Camellia sinensis, Rhodiola rosea, and Koelreuteria henryi exhibited good tyrosinase inhibitory activities and potent anti-lipoxygenase activities. As Koelreuteria henryi had notable significant inhibitory activities towards xanthine oxidase, tyrosinase, and lipoxygenase, it was further tested with tyrosinase and HPLC-DAD. The results from this part of the study revealed that the more powerful the antioxidant capability of the extracted component, the greater the decrease in peak height obtained after reacting with tyrosinase. Additional studies are warranted to further characterize the compounds responsible for the antioxidant properties of the examined extracts.

  13. Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

    Science.gov (United States)

    Wang, Ke; Li, Nan; Zhang, Jing; Zhang, Zhiqi; Dang, Fuquan

    2017-01-15

    In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H 2 O 2 , substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H 2 O 2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H 2 O 2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination

    Directory of Open Access Journals (Sweden)

    Wen-Yan Li

    2017-01-01

    Full Text Available Seed germination is a complicated biological process that requires regulation through various enzymatic and non-enzymatic mechanisms. Although it has been recognized that reactive oxygen species (ROS regulate radicle emergence and root elongation in a non-enzymatic manner during dicot seed germination, the role of ROS in monocot seed germination remains unknown. NADPH oxidases (NOXs are the major ROS producers in plants; however, whether and how NOXs regulate rice seed germination through ROS generation remains unclear. Here, we report that diphenyleneiodinium (DPI, a specific NOX inhibitor, potently inhibited embryo and seedling growth—especially that of the radicle and of root elongation—in a dose-dependent manner. Notably, the DPI-mediated inhibition of radicle and root growth could be eliminated by transferring seedlings from DPI to water. Furthermore, ROS production/accumulation during rice seed germination was quantified via histochemistry. Superoxide radicals (O2−, hydrogen peroxide (H2O2 and hydroxyl radicals (•OH accumulated steadily in the coleorhiza, radicle and seedling root of germinating rice seeds. Expression profiles of the nine typical NOX genes were also investigated. According to quantitative PCR, OsNOX5, 7 and 9 were expressed relatively higher. When seeds were incubated in water, OsNOX5 expression progressively increased in the embryo from 12 to 48 h, whereas OsNOX7 and 9 expressions increased from 12 to 24 h and decreased thereafter. As expected, DPI inhibits the expression at predetermined time points for each of these genes. Taken together, these results suggest that ROS produced by NOXs are involved in radicle and root elongation during rice seed germination, and OsNOX5, 7 and 9 could play crucial roles in rice seed germination. These findings will facilitate further studies of the roles of ROS generated by NOXs during seed germination and seedling establishment and also provide valuable information for the

  15. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei, E-mail: hmliu2004@126.com [Huazhong University of Science and Technology, School of Chemistry and Chemical Engineering (China)

    2016-03-15

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na{sub 2}SeO{sub 3} as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K{sub m}) values and maximal reaction velocity (V{sub max}) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  16. Cambogin exerts anti-proliferative and pro-apoptotic effects on breast adenocarcinoma through the induction of NADPH oxidase 1 and the alteration of mitochondrial morphology and dynamics.

    Science.gov (United States)

    Shen, Kaikai; Lu, Fangfang; Xie, Jianling; Wu, Minfeng; Cai, Bo; Liu, Yurong; Zhang, Hong; Tan, Hongsheng; Pan, Yingyi; Xu, Hongxi

    2016-08-02

    Cambogin, a bioactive polycyclic polyprenylated acylphoroglucinol (PPAP) derived from the Garcinia genus, possesses proapoptotic effect in medulloblastoma and breast cancer cells. We have previously demonstrated that the proapoptotic effect of cambogin is driven by the production of reactive oxygen species (ROS). Here we have shown that the inhibitory effect of cambogin on cell proliferation is associated with the loss of mitochondrial transmembrane potential (ΔΨm) and mitochondrial fragmentation. Cambogin also promotes the mutual complex formation of the membrane-bound subunit p22phox of NADPH oxidase 1 (NOX1), as well as the phosphorylation of the cytosolic subunit p47phox, subsequently enhancing membrane-bound NOX1 activity, which leads to increases in intracellular and mitochondrial levels of O2.- and H2O2. Pharmacological inhibition of NOX1 using apocynin (pan-NOX inhibitor), ML171 (NOX1 inhibitor) or siRNA against NOX1 prevents the increases in O2.- and H2O2 levels and the anti-proliferative effect of cambogin. Antioxidants, including SOD (superoxide dismutase), CAT (catalase) and EUK-8, are also able to restore cell viability in the presence of cambogin. Besides, cambogin increases the dissociation of thioredoxin-1 (Trx1) from ASK1, switching the inactive form of ASK1 to the active kinase, subsequently leads to the phosphorylation of JNK/SAPK, which is abolished upon ML171 treatment. The proapoptotic effect of cambogin in breast cancer cells is also aggravated upon knocking down Trx1 in MCF-7 cells. Taken in conjunction, these data indicate that the anti-proliferative and pro-apoptotic effect of cambogin is mediated via inducing NOX1-dependent ROS production and the dissociation of ASK1 and Trx1.

  17. Axotomy increases NADPH-diaphorase activity in the dorsal root ganglia and lumbar spinal cord of the turtle Trachemys dorbigni

    OpenAIRE

    Partata,W.A.; Krepsky,A.M.R.; Marques,M.; Achaval,M.

    1999-01-01

    Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spina...

  18. Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

    Science.gov (United States)

    Fraser, D A; Hessvik, N P; Nikolić, N; Aas, V; Hanssen, K F; Bøhn, S K; Thoresen, G H; Rustan, A C

    2012-07-01

    The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 μM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 μM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 μM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

  19. Resveratrol Protects Dopamine Neurons Against Lipopolysaccharide-Induced Neurotoxicity through Its Anti-Inflammatory Actions

    Science.gov (United States)

    Zhang, Feng; Shi, Jing-Shan; Zhou, Hui; Wilson, Belinda; Hong, Jau-Shyong

    2010-01-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by a progressive loss of dopamine (DA) neurons in the substantia nigra. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. Resveratrol, a nonflavonoid polyphenol naturally found in red wine and grapes, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. Although recent studies have shown that resveratrol provided neuroprotective effects against ischemia, seizure, and neurodegenerative disorders, the mechanisms underlying its beneficial effects on dopaminergic neurodegeneration are poorly defined. In this study, rat primary midbrain neuron-glia cultures were used to elucidate the molecular mechanisms underlying resveratrol-mediated neuroprotection. The results clearly demonstrated that resveratrol protected DA neurons against lipopolysaccharide (LPS)-induced neurotoxicity in concentration- and time-dependent manners through the inhibition of microglial activation and the subsequent reduction of proinflammatory factor release. Mechanistically, resveratrol-mediated neuroprotection was attributed to the inhibition of NADPH oxidase. This conclusion is supported by the following observations. First, resveratrol reduced NADPH oxidase-mediated generation of reactive oxygen species. Second, LPS-induced translocation of NADPH oxidase cytosolic subunit p47 to the cell membrane was significantly attenuated by resveratrol. Third and most importantly, resveratrol failed to exhibit neuroprotection in cultures from NADPH oxidase-deficient mice. Furthermore, this neuroprotection was also related to an attenuation of the activation of mitogen-activated protein kinases and nuclear factor-κB signaling pathways in microglia. These findings suggest that resveratrol exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and NADPH oxidase may be a major player

  20. Effect of GLP-1 on the expression of NADPH oxidase subunits in the kidney of type 1 diabetic rats

    Directory of Open Access Journals (Sweden)

    Jin-jin LIU

    2013-09-01

    Full Text Available Objective To observe the effect of exenatide, a glucagon-like peptide-1 (GLP-1 receptor agonist, on the expression of NADPH oxidase subunits NOX4 and p22phox and connective tissue growth factor (CTGF in the kidney of streptozotocin (STZ-induced type 1 diabetic rats, and explore the protective effects and mechanisms of exenatide on the kidney of diabetic rats. Methods Thirty male Sprague-Dawley (SD rats were divided into control group (group A, n=7 and diabetic model group (n=23. Type 1 diabetic model was reproduced by intraperitoneal injection of streptozotocin. It was successful in 19 rats. Diabetic rats were randomly divided into diabetic control group (group B, n=10 and diabetic with treatment of exenatide group (group C, n=9. Rats in group C were injected subcutaneously with exenatide in dose of 5μg/kg twice daily. Rats in group A and B were given equivalent volume of normal saline by subcutaneous injection. All rats were sacrificed after eight weeks. The mRNA expression of renal p22phox and NOX4 were detected by real-time fluorescence quantitative PCR. The protein expression of CTGF was detected by immunohistochemical staining. Results The levels of blood glucose, lipids, creatinine, and urea nitrogen, the albumin excretion rate, kidney index, the mRNA expressions of renal NOX4 and p22phox, and the protein expression of renal CTGF were significantly increased in group B compared with that in group A (P0.05. Conclusion Exenatide can decrease the expressions of renal NOX4, p22phox and CTGF, decline the index of urinary protein, and alleviate the kidney hypertrophy in type 1 diabetic rats, implying that exenatide exerted a protective effect on the kidney.

  1. Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC

    Directory of Open Access Journals (Sweden)

    Zeynab Nayernia

    2017-10-01

    Full Text Available There is emerging evidence for the involvement of reactive oxygen species (ROS in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC. High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST, and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.

  2. Activation of neuronal nitric oxide synthase in cerebellum of chronic hepatic encephalopathy rats is associated with up-regulation of NADPH-producing pathway.

    Science.gov (United States)

    Singh, Santosh; Trigun, Surendra K

    2010-09-01

    Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH-diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.

  3. Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22phox expression

    International Nuclear Information System (INIS)

    Wang, Chaoyun; He, Yanhao; Yang, Ming; Sun, Hongliu; Zhang, Shuping; Wang, Chunhua

    2013-01-01

    Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels of target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22 phox , increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation. - Highlights: • Angiotensin II depresses mitochondria physiological function. • Angiotensin II activates NADPH oxidase via up-regulating expresion of p22 phox . • Bcl-2 plays a pivotal role in improving mitochondria function and regulates ROS level. • Inhibitor of Bcl-2 promotes angiotensin II mediated HUVEC injury. • SYB attenuates angiotensin II mediated HUVEC injury via up regulating Bcl-2 expression

  4. Myeloperoxidase-Dependent LDL Modifications in Bloodstream Are Mainly Predicted by Angiotensin II, Adiponectin, and Myeloperoxidase Activity: A Cross-Sectional Study in Men

    Directory of Open Access Journals (Sweden)

    Karim Zouaoui Boudjeltia

    2013-01-01

    Full Text Available The present paradigm of atherogenesis proposes that low density lipoproteins (LDLs are trapped in subendothelial space of the vascular wall where they are oxidized. Previously, we showed that oxidation is not restricted to the subendothelial location. Myeloperoxidase (MPO, an enzyme secreted by neutrophils and macrophages, can modify LDL (Mox-LDL at the surface of endothelial cells. In addition we observed that the activation of the endothelial cells by angiotensin II amplifies this process. We suggested that induction of the NADPH oxidase complex was a major step in the oxidative process. Based on these data, we asked whether there was an independent association, in 121 patients, between NADPH oxidase modulators, such as angiotensin II, adiponectin, and levels of circulating Mox-LDL. Our observations suggest that the combination of blood angiotensin II, MPO activity, and adiponectin explains, at least partially, serum Mox-LDL levels.

  5. Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

    Science.gov (United States)

    Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

    2008-06-01

    We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

  6. Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

    Science.gov (United States)

    Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

    1991-01-01

    Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

  7. Class IA phosphoinositide 3-kinase β and δ regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae.

    Science.gov (United States)

    Boyle, Keith B; Gyori, David; Sindrilaru, Anca; Scharffetter-Kochanek, Karin; Taylor, Philip R; Mócsai, Attila; Stephens, Len R; Hawkins, Phillip T

    2011-03-01

    An effective immune response to the ubiquitous fungus Aspergillus fumigatus is dependent upon production of reactive oxygen species (ROS) by the NADPH oxidase. This is evidenced by the acute sensitivity of oxidase-deficient humans and mice to invasive aspergillosis. Neutrophils are recruited to the lungs shortly postinfection and respond by phagocytosing conidia and mediating extracellular killing of germinated hyphae in a ROS-dependent manner. However, the signaling mechanisms regulating the generation of ROS in response to hyphae are poorly understood. PI3Ks are important regulators of numerous cellular processes, with much recent work describing unique roles for the different class I PI3K isoforms. We showed by live-cell imaging that the lipid products of class I PI3Ks accumulated at the hyphal-bound neutrophil plasma membrane. Further, we used pharmacological and genetic approaches to demonstrate essential, but overlapping, roles for PI3Kβ and PI3Kδ in the ROS and spreading responses of murine neutrophils to Aspergillus hyphae. Hyphal-induced ROS responses were substantially inhibited by deletion of the common β2-integrin subunit CD18, with only a minor, redundant role for Dectin-1. However, addition of soluble algal glucans plus the genetic deletion of CD18 were required to significantly inhibit activation of the PI3K-effector protein kinase B. Hyphal ROS responses were also totally dependent on the presence of Syk, but not its ITAM-containing adaptor proteins FcRγ or DAP12, and the Vav family of Rac-guanine nucleotide exchange factors. These results start to define the signaling network controlling neutrophil ROS responses to A. fumigatus hyphae.

  8. Glucose oxidase variants with improved properities

    OpenAIRE

    Fischer, Rainer; Ostafe, Raluca; Prodanovic, Radivoje

    2014-01-01

    Source: WO14173822A3 [EN] The technology provided herein relates to novel variants of microbial glucose oxidase with improved properties, more specifically to polypeptides having glucose oxidase activity as their major enzymatic activity; to nucleic acid molecules encoding said glucose oxidases; vectors and host cells containing the nucleic acids and methods for producing the glucose oxidase; compositions comprising said glucose oxidase; methods for the preparation and production of such enzy...

  9. Pro-inflammatory effects of interleukin-17A on vascular smooth muscle cells involve NAD(P)H- oxidase derived reactive oxygen species.

    Science.gov (United States)

    Pietrowski, Eweline; Bender, Bianca; Huppert, Jula; White, Robin; Luhmann, Heiko J; Kuhlmann, Christoph R W

    2011-01-01

    T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2'7'-dichlorodihydrofluorescein (H(2)DCF) in primary murine VSMC. IL-17A induced an increase in H(2)DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting Nox2. The p38-MAPK inhibitors SB203580 and SB202190 dose-dependently reduced the IL-17A induced ROS production. The IL-17A induced release of the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and MCP-1 from VSMC, as detected by the Luminex technology, was completely abolished by NAD(P)H-oxidase inhibition. Taken together, our data indicate that IL-17A causes the NAD(P)H-oxidase dependent generation of ROS leading to a pro-inflammatory activation of VSMC. Copyright © 2010 S. Karger AG, Basel.

  10. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  11. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    Science.gov (United States)

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. WO3/Pt nanoparticles are NADPH oxidase biomimetics that mimic effector cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Clark, Andrea J; Coury, Emma L; Meilhac, Alexandra M; Petty, Howard R

    2016-01-01

    To provide a means of delivering an artificial immune effector cell-like attack on tumor cells, we report the tumoricidal ability of inorganic WO 3 /Pt nanoparticles that mimic a leukocyte’s functional abilities. These nanoparticles route electrons from organic structures and electron carriers to form hydroxyl radicals within tumor cells. During visible light exposure, WO 3 /Pt nanoparticles manufacture hydroxyl radicals, degrade organic compounds, use NADPH, trigger lipid peroxidation, promote lysosomal membrane disruption, promote the loss of reduced glutathione, and activate apoptosis. In a model of advanced breast cancer metastasis to the eye’s anterior chamber, we show that WO 3 /Pt nanoparticles prolong the survival of 4T1 tumor-bearing Balb/c mice. This new generation of inorganic photosensitizers do not photobleach, and therefore should provide an important therapeutic advance in photodynamic therapy. As biomimetic nanoparticles destroy targeted cells, they may be useful in treating ocular and other forms of cancer. (paper)

  13. WO3/Pt nanoparticles are NADPH oxidase biomimetics that mimic effector cells in vitro and in vivo

    Science.gov (United States)

    Clark, Andrea J.; Coury, Emma L.; Meilhac, Alexandra M.; Petty, Howard R.

    2016-02-01

    To provide a means of delivering an artificial immune effector cell-like attack on tumor cells, we report the tumoricidal ability of inorganic WO3/Pt nanoparticles that mimic a leukocyte’s functional abilities. These nanoparticles route electrons from organic structures and electron carriers to form hydroxyl radicals within tumor cells. During visible light exposure, WO3/Pt nanoparticles manufacture hydroxyl radicals, degrade organic compounds, use NADPH, trigger lipid peroxidation, promote lysosomal membrane disruption, promote the loss of reduced glutathione, and activate apoptosis. In a model of advanced breast cancer metastasis to the eye’s anterior chamber, we show that WO3/Pt nanoparticles prolong the survival of 4T1 tumor-bearing Balb/c mice. This new generation of inorganic photosensitizers do not photobleach, and therefore should provide an important therapeutic advance in photodynamic therapy. As biomimetic nanoparticles destroy targeted cells, they may be useful in treating ocular and other forms of cancer.

  14. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    International Nuclear Information System (INIS)

    Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

    2012-01-01

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

  15. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  16. Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

    International Nuclear Information System (INIS)

    Arana, Lide; Gangoiti, Patricia; Ouro, Alberto; Rivera, Io-Guané; Ordoñez, Marta; Trueba, Miguel; Lankalapalli, Ravi S.; Bittman, Robert; Gomez-Muñoz, Antonio

    2012-01-01

    We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A 2 and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA 2 -α in this pathway. -- Highlights: ► Ceramide 1-phosphate (C1P) stimulates reactive oxygen species (ROS) formation. ► The enzyme responsible for ROS generation by C1P in macrophages is NADPH oxidase. ► NADPH oxidase lies downstream of cPLA 2 -α and PKC-α in this pathway. ► ROS generation is essential for the stimulation of macrophage proliferation by C1P.

  17. Voltage-gated proton channel is expressed on phagosomes

    International Nuclear Information System (INIS)

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-01-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  18. MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Simões, Maylla Ronacher, E-mail: yllars@hotmail.com [Dept. of Physiological Sciences, Federal University of Espirito Santo, Vitória, ES CEP 29040-091 (Brazil); Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), Madrid (Spain); Aguado, Andrea [Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), Madrid (Spain); Fiorim, Jonaína; Silveira, Edna Aparecida; Azevedo, Bruna Fernandes; Toscano, Cindy Medice [Dept. of Physiological Sciences, Federal University of Espirito Santo, Vitória, ES CEP 29040-091 (Brazil); Zhenyukh, Olha; Briones, Ana María [Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), Madrid (Spain); Alonso, María Jesús [Dept. of Biochemistry, Physiology and Molecular Genetics, Universidad Rey Juan Carlos, Alcorcón (Spain); Vassallo, Dalton Valentim [Dept. of Physiological Sciences, Federal University of Espirito Santo, Vitória, ES CEP 29040-091 (Brazil); Health Science Center of Vitória-EMESCAM, Vitória, ES CEP 29045-402 (Brazil); Salaices, Mercedes, E-mail: mercedes.salaices@uam.es [Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), Madrid (Spain)

    2015-03-01

    Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20 μg/dL) were used. Lead blood levels of treated rats attained 21.7 ± 2.38 μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension. - Highlights: • Lead-exposure increases oxidative stress, COX-2 expression and vascular reactivity. • Lead exposure activates MAPK signaling pathway. • ROS and COX-2 activation by

  19. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

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

    Science.gov (United States)

    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.

  1. Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia

    Science.gov (United States)

    Laidlaw, Kamilla M.E.; Berhan, Samuel; Liu, Suhu; Silvestri, Giovannino; Holyoake, Tessa L.; Frank, David A.; Aggarwal, Bharat; Bonner, Michael Y.; Perrotti, Danilo

    2016-01-01

    The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation. PMID:27438151

  2. Superoxide Mediates Depressive Effects Induced by Hydrogen Sulfide in Rostral Ventrolateral Medulla of Spontaneously Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Haiyun Yu

    2015-01-01

    Full Text Available Hydrogen sulfide (H2S plays a crucial role in the regulation of blood pressure and oxidative stress. In the present study, we tested the hypothesis that H2S exerts its cardiovascular effects by reducing oxidative stress via inhibition of NADPH oxidase activity in the rostral ventrolateral medulla (RVLM. We examined cell distributions of cystathionine-β-synthase (CBS and effects of H2S on reactive oxygen species (ROS and mean arterial blood pressure (MAP in spontaneously hypertensive rats (SHRs. We found that CBS was expressed in neurons of the RVLM, and the expression was lower in SHRs than in Wistar-Kyoto rats. Microinjection of NaHS (H2S donor, S-adenosyl-l-methionine (SAM, a CBS agonist, or Apocynin (NADPH oxidase inhibitor into the RVLM reduced the ROS level, NADPH oxidase activity, and MAP, whereas microinjection of hydroxylamine hydrochloride (HA, a CBS inhibitor increased MAP. Furthermore, intracerebroventricular infusion of NaHS inhibited phosphorylation of p47phox, a key step of NADPH oxidase activation. Since decreasing ROS level in the RVLM reduces MAP and heart rate and increasing H2S reduces ROS production, we conclude that H2S exerts an antihypertensive effect via suppressing ROS production. H2S, as an antioxidant, may be a potential target for cardiovascular diseases.

  3. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    Science.gov (United States)

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  4. [The distribution of NADPH-diaphorase and neuronal no synthase in rat medulla oblongata nuclei].

    Science.gov (United States)

    Chertok, V M; Kotsuba, A E

    2013-01-01

    The distribution of nitroxide ergic neurons in the medulla oblongata nuclei in Wistar rats (n = 8) was studied histochemically (NADPH-diaphorase) and using immunohistochemistry with an antiserum against neuronal form of nitric oxide synthase (nNOS). NADPH-diaphorase activity was found in large and small neurons of the sensory, autonomic and motor nuclei. The latter were especially rich in the cells demonstrating the activity of the enzyme. Unlike NADPH-diaphorase, nNOS in the corresponding nuclei was always detected in the fewer number of neurons, predominantly of small sizes. The sensory nuclei (nucleus of solitary tract, reticular parvocellular and lateral nuclei, spinal nucleus of the trigeminal nerve) contained 1.5-3 times more nNOS neurons than in motor nuclei. In some nuclei (nucleus ambiguus, hypoglossal nerve nucleus), containing numerous NADPH-diaphorase-positive neurons, immunoreactive cells were particularly rare.

  5. Axotomy increases NADPH-diaphorase activity in the dorsal root ganglia and lumbar spinal cord of the turtle Trachemys dorbigni

    Directory of Open Access Journals (Sweden)

    Partata W.A.

    1999-01-01

    Full Text Available Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.

  6. Axotomy increases NADPH-diaphorase activity in the dorsal root ganglia and lumbar spinal cord of the turtle Trachemys dorbigni.

    Science.gov (United States)

    Partata, W A; Krepsky, A M; Marques, M; Achaval, M

    1999-04-01

    Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.

  7. Circadian and Metabolic Perspectives in the Role Played by NADPH in Cancer

    Directory of Open Access Journals (Sweden)

    Isabel Méndez

    2018-03-01

    Full Text Available Physiological activity in healthy conditions requires a coordinated interaction between the molecular circadian clock and the network of biochemical pathways. An important metabolic parameter in the interface between these two entities is the redox state. Among the redox coenzymes that regulate the fluxes of enzymatic reactions is the NADP+/NADPH pair. Indeed, the main biosynthetic pathways need NADPH to serve as an electron donor for cellular anabolic transformations. The existence of a metabolic circadian clock is well established, and it was first identified in mammalian red blood cells. The metabolic circadian clock is independent of transcriptional activity and is sustained by the enzymatic complex peroxiredoxin/thioredoxin/NADPH. This complex shows 24-h redox fluctuations metabolizing H2O2 in various tissues and species (fungi, insects, and mammals. Although this NADPH-sensitive metabolic clock is autonomous in erythrocytes that lack a nucleus, it functions in concert with the transcriptional circadian clock in other cell types to accomplish the task of timing cellular physiology. During carcinogenesis, circadian alterations influence cell cycle onset and promote tumoral growth. These alterations also deregulate cellular energetics through a process known as aerobic glycolysis, or the Warburg effect. The Warburg effect is a typical response of cancer cells in which the metabolism turns into glycolysis even in the presence of functional mitochondria. This alteration has been interpreted as a cellular strategy to increase biomass during cancer, and one of its main factors is the availability of NADPH. This minireview explores the potential role of NADPH as a circadian and cancer-promoting metabolite.

  8. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  9. Rac-1 as a new therapeutic target in cerebro- and cardio-vascular diseases.

    Science.gov (United States)

    Carrizzo, Albino; Forte, Maurizio; Lembo, Maria; Formisano, Luigi; Puca, Annibale A; Vecchione, Carmine

    2014-01-01

    Growing evidence indicates that overproduction of reactive oxygen species (ROS) plays a prominent role in the development of cardio- and cerebro-vascular diseases. Among the mechanisms identified to produce oxidative stress in the vascular wall, those mediated by membrane-bound NAD(P)H oxidases represent a major one. NAD(P)H oxidases are a family of enzymes that generate ROS both in phagocytic and non-phagocytic cell types. Vascular NAD(P)H oxidase contains the membrane-bound subunits Nox1, Nox2 (gp91phox), Nox4 and p22phox, the catalytic site of the oxidase, and the cytosolic components p47phox and p67phox. Rac1 (Ras-related C3 botulinum toxin substrate1) is a small GTPase essential for the assembly and activation of NADPH oxidase. Several molecular and cellular studies have reported the involvement of Rac1 in different cardiovascular pathologies, such as vascular smooth muscle proliferation, cardiomyocyte hypertrophy, endothelial cell shape change, atherosclerosis and endothelial dysfunction in hypertension. In addition, increased activation of NADPH oxidase by Rac1 has been reported in animals and humans after myocardial infarction and heart failure. The Rac1/NADPH pathway has also been found involved in different pathologies of the cerebral district, such as ischemic stroke, cognitive impairment, subaracnoid hemorrhage and neuronal oxidative damage typical of several neurodegenerative disorders. In addition, thrombotic events are an important step in the onset of cardio- and cerebrovascular diseases. Rac1 has been found involved also in platelet activation, inducing actin polymerization and lamellipodia formation, which are necessary steps for platelet aggregation. Taken together, the evidence candidates Rac1 as a new pharmacological target of cardiovascular and cerebrovascular diseases. Although the involvement of Rac1 in the beneficial pleiotropic effects of drugs such as statins is well known, and the onset of numerous side effects has raised concern for the

  10. Intake and time dependence of blueberry flavonoid-induced improvements in vascular function: a randomized, controlled, double-blind, crossover intervention study with mechanistic insights into biological activity.

    Science.gov (United States)

    Rodriguez-Mateos, Ana; Rendeiro, Catarina; Bergillos-Meca, Triana; Tabatabaee, Setareh; George, Trevor W; Heiss, Christian; Spencer, Jeremy Pe

    2013-11-01

    There are very limited data regarding the effects of blueberry flavonoid intake on vascular function in healthy humans. We investigated the impact of blueberry flavonoid intake on endothelial function in healthy men and assessed potential mechanisms of action by the assessment of circulating metabolites and neutrophil NADPH oxidase activity. Two randomized, controlled, double-blind, crossover human-intervention trials were conducted with 21 healthy men. Initially, the impact of blueberry flavonoid intake on flow-mediated dilation (FMD) and polyphenol absorption and metabolism was assessed at baseline and 1, 2, 4, and 6 h after consumption of blueberry containing 766, 1278, and 1791 mg total blueberry polyphenols or a macronutrient- and micronutrient-matched control drink (0 mg total blueberry polyphenols). Second, an intake-dependence study was conducted (from baseline to 1 h) with 319, 637, 766, 1278, and 1791 mg total blueberry polyphenols and a control. We observed a biphasic time-dependent increase in FMD, with significant increases at 1-2 and 6 h after consumption of blueberry polyphenols. No significant intake-dependence was observed between 766 and 1791 mg. However, at 1 h after consumption, FMD increased dose dependently to ≤766 mg total blueberry polyphenol intake, after which FMD plateaued. Increases in FMD were closely linked to increases in circulating metabolites and by decreases in neutrophil NADPH oxidase activity at 1-2 and 6 h. Blueberry intake acutely improves vascular function in healthy men in a time- and intake-dependent manner. These benefits may be mechanistically linked to the actions of circulating phenolic metabolites on neutrophil NADPH oxidase activity. This trial was registered at clinicaltrials.gov as NCT01292954 and NCT01829542.

  11. Endogenous mechanisms of reactive oxygen species (ROS generation

    Directory of Open Access Journals (Sweden)

    Agata Sarniak

    2016-11-01

    Full Text Available The main cellular source of reactive oxygen species (ROS is mitochondrial respiratory chain and active NADPH responsible for “respiratory burst” of phagocytes. Whatsmore ROS are produced in endoplasmic reticulum, peroxisomes, with the participation of xanthine and endothelial oxidase and during autoxidation process of small molecules. Mitochondrial respiratory chain is the main cellular source of ROS. It is considered that in aerobic organisms ROS are mainly formed during normal oxygen metabolism, as byproducts of oxidative phosphorylation, during the synthesis of ATP. The intermembranous phagocyte enzyme – activated NADPH oxidase, responsible for the “respiratory burst” of phagocytes, which is another source of ROS, plays an important role in defense of organism against infections.The aim of this article is to resume actuall knowledge about structure and function of the mitochondrial electron transport chain in which ROS are the byproducts and about NADPH oxidase as well as the function of each of its components in the “respiratory burst” of phagocytes.

  12. Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery

    Directory of Open Access Journals (Sweden)

    Guillermo Bodega

    2018-01-01

    Full Text Available In a previous study, we demonstrated that endothelial microvesicles (eMVs have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(PH that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(PH synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

  13. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    Science.gov (United States)

    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

  14. Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

    Directory of Open Access Journals (Sweden)

    Tao Zhan

    Full Text Available 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 Arg(51 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.

  15. Pivotal Advance: Eosinophilia in the MES rat strain is caused by a loss-of-function mutation in the gene for cytochrome b(-245), alpha polypeptide (Cyba).

    Science.gov (United States)

    Mori, Masayuki; Li, Guixin; Hashimoto, Maiko; Nishio, Ayako; Tomozawa, Hiroshi; Suzuki, Nobuyoshi; Usami, Shin-ichi; Higuchi, Keiichi; Matsumoto, Kiyoshi

    2009-09-01

    MES is a rat strain that spontaneously develops severe blood eosinophilia as a hereditary trait. Herein, we report that eosinophilia in MES rats is caused by a loss-of-function mutation in the gene for cytochrome b(-245), alpha polypeptide (Cyba; also known as p22(phox)), which is an essential component of the superoxide-generating NADPH oxidase complex. The MES rat has a deletion of four nucleotides, including the 5' splice donor GpT of intron 4 of the Cyba gene. As a consequence of the deletion, a 51-nucleotide sequence of intron 4 is incorporated into the Cyba transcripts. Leukocytes from the MES strain lack both CYBA protein and NADPH oxidase activity. Nevertheless, unlike patients with chronic granulomatous disease, who suffer from infections with pathogens due to similar genetic defects in NADPH oxidase, MES rats retain normal innate immune defense against Staphylococcus aureus infection. This is due to large quantities of peritoneal eosinophils in MES rats, which phagocytose and kill the bacteria. MES rat has a balance defect due to impaired formation of otoconia in the utricles and saccules. Eosinophilia of the MES rat was normalized by introduction of a normal Cyba transgene. The mechanisms by which impairment of NADPH oxidase leads to eosinophilia in the MES rat are elusive. However, our study highlights the essential role of NADPH oxidase in homeostatic regulation of innate immunity beyond conventional microbicidial functions.

  16. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

    International Nuclear Information System (INIS)

    Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

    1978-01-01

    The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

  17. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → 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 2 O 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.

  20. NADPH oxidases in Microglia oxidant production

    DEFF Research Database (Denmark)

    Haslund-Vinding, J; McBean, G; Jaquet, V

    2017-01-01

    inhibitors. Finally, we review the recent literature on NOX and other sources of ROS that are involved in activation of the inflammasome and discuss the potential influence of microglia-derived oxidants on neurogenesis, neural differentiation and culling of surplus progenitor cells. The degree to which...

  1. Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation.

    Science.gov (United States)

    Shi, Long; Chen, Hongmei; Yu, Xiaoming; Wu, Xinyi

    2013-11-01

    Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.

  2. Introducing extra NADPH consumption ability significantly increases the photosynthetic efficiency and biomass production of cyanobacteria.

    Science.gov (United States)

    Zhou, Jie; Zhang, Fuliang; Meng, Hengkai; Zhang, Yanping; Li, Yin

    2016-11-01

    Increasing photosynthetic efficiency is crucial to increasing biomass production to meet the growing demands for food and energy. Previous theoretical arithmetic analysis suggests that the light reactions and dark reactions are imperfectly coupled due to shortage of ATP supply, or accumulation of NADPH. Here we hypothesized that solely increasing NADPH consumption might improve the coupling of light reactions and dark reactions, thereby increasing the photosynthetic efficiency and biomass production. To test this hypothesis, an NADPH consumption pathway was constructed in cyanobacterium Synechocystis sp. PCC 6803. The resulting extra NADPH-consuming mutant grew much faster and achieved a higher biomass concentration. Analyses of photosynthesis characteristics showed the activities of photosystem II and photosystem I and the light saturation point of the NADPH-consuming mutant all significantly increased. Thus, we demonstrated that introducing extra NADPH consumption ability is a promising strategy to increase photosynthetic efficiency and to enable utilization of high-intensity lights. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Eun-Mi Noh

    2016-01-01

    Full Text Available Reactive oxygen species (ROS play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3 metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

  4. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection

    Science.gov (United States)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

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

    Science.gov (United States)

    Yeşiller, 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

    Science.gov (United States)

    Elmasry, Khaled; Ibrahim, Ahmed S; Saleh, Heba; Elsherbiny, Nehal; Elshafey, Sally; Hussein, Khaled A; Al-Shabrawey, Mohamed

    2018-05-01

    Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo -/- ); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2 -/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca 2+ ) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO

  7. Season-controlled changes in biochemical constituents and oxidase enzyme activities in tomato (Lycopersicon esculentum Mill.).

    Science.gov (United States)

    Sen, Supatra; Mukherji, S

    2009-07-01

    Season-controlled changes in biochemical constituents viz. carotenoids (carotene and xanthophyll) and pectic substances along with IAA-oxidase and polyphenol oxidase (PPO) enzyme activities were estimated/assayed in leaves of Lycopersicon esculentum Mill. (tomato) in two developmental stages--pre-flowering (35 days after sowing) and post-flowering (75 days after sowing) in three different seasons--summer rainy and winter Carotenoid content along with pectic substances were highest in winter and declined significantly in summer followed by rainy i.e. winter > summer > rainy. Carotenoid content was significantly higher in the pre-flowering as compared to post-flowering in all three seasons while pectic substances increased in the post-flowering as compared to pre-flowering throughout the annual cycle. IAA oxidase and PPO enzyme activities were enhanced in rainy and decreased sharply in summer and winter i.e. rainy > summer > winter. Both the enzymes exhibited higher activity in the post-flowering stage as compared to pre-flowering in all three seasons. These results indicate winter to be the most favourable season for tomato plants while rainy season environmental conditions prove to be unfavourable (stressful) with diminished content of carotenoid and pectic substances and low activities of IAA oxidase and PPO, ultimately leading to poor growth and productivity.

  8. Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

    Directory of Open Access Journals (Sweden)

    Wei Chin-Chuan

    2012-04-01

    Full Text Available Abstract Background Superoxide generated by non-phagocytic NADPH oxidases (NOXs is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD and its calcium-binding knockout mutants. Results The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q and NCaBD(E63Q showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD. Conclusions We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. Graphical abstract

  9. Engineering an NADPH/NADPRedox Biosensor in Yeast

    DEFF Research Database (Denmark)

    Zhang, Jie; Sonnenschein, Nikolaus; Pihl, Thomas Peter Boye

    2016-01-01

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biote......Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science...... in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon...... NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP+ ratios from mixed cell populations. We show...

  10. Involvement of Polyamines in the Chilling Tolerance of Cucumber Cultivars

    Science.gov (United States)

    Shen, Wenyun; Nada, Kazuyoshi; Tachibana, Shoji

    2000-01-01

    The possible involvement of polyamines (PAs) in the chilling tolerance of cucumber (Cucumis sativus L. cv Jinchun No. 3 and cv Suyo) was investigated. Plants with the first expanded leaves were exposed to 3°C or 15°C in the dark for 24 h (chilling), and then transferred to 28°C/22°C under a 12-h photoperiod for another 24 h (rewarming). Chilling-tolerant cv Jinchun No. 3 showed a marked increase of free spermidine (Spd) in leaves, once during chilling and again during rewarming. Putrescine increased significantly during rewarming, but the increase of spermine was slight. Any of these PAs did not increase in chilling-sensitive cv Suyo during either period. PA-biosynthetic enzyme activities appear to mediate these differences between cultivars. Pretreatment of Spd to cv Suyo prevented chill-induced increases in the contents of hydrogen peroxide in leaves and activities of NADPH oxidases and NADPH-dependent superoxide generation in microsomes and alleviated chilling injury. Pretreatment of methylglyoxal-bis-(guanylhydrazone), a PA biosynthesis inhibitor, to chilled cv Jinchun No. 3 prevented Spd increase and enhanced microsomal NADPH oxidase activity and chilling injury. The results suggest that Spd plays important roles in chilling tolerance of cucumber, probably through prevention of chill-induced activation of NADPH oxidases in microsomes. PMID:10982456

  11. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2013-12-01

    Full Text Available This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%, DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, respectively, which after desalting was 15.52 and 12.05 U mg-1, and after ion exchange and gel filtration chromatography was 29.09 - 62 and 25.72 - 59.37 U mg-1 for A. niger and P. notatum, respectively. The antimicrobial activity was determined by disc diffusion method against selected microbial strains where glucose oxidase from A. niger showed anti-bacterial activity, while no fungicidal effects were shown by both A. niger and P. notatum glucose oxidases.

  12. Frontline Science: HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients who survive septic shock.

    Science.gov (United States)

    Grégoire, Murielle; Tadié, Jean-Marc; Uhel, Fabrice; Gacouin, Arnaud; Piau, Caroline; Bone, Nathaniel; Le Tulzo, Yves; Abraham, Edward; Tarte, Karin; Zmijewski, Jaroslaw W

    2017-06-01

    Sepsis is accompanied by the initial activation of proinflammatory pathways and long-lasting immunosuppression that appears to contribute to late-occurring mortality. Although high-mobility group box 1 (HMGB1) is involved in many aspects of inflammation, its role in sepsis-induced immune suppression remains unclear. In this study, we examined HMGB1's contribution to neutrophil NADPH oxidase activity dysfunction and associated neutrophil-dependent bacterial clearance in mice subjected to sepsis and in patients who survive septic shock. Using a murine model of polymicrobial septic peritonitis, we demonstrated that treatment with anti-HMGB1 Ab significantly diminished sepsis-induced dysfunction of neutrophil NADPH oxidase activity. In a subsequent set of experiments, we found that blocking HMGB1 preserved the ability of neutrophils from patients recovering from septic shock to activate NADPH oxidase. Taken together, our data suggest that HMGB1 accumulation in the late phase of sepsis plays a specific role in the development of postsepsis immunosuppression and specifically affects neutrophil-dependent antibacterial defense mechanisms. Thus, blocking HMGB1 may be a promising therapeutic intervention to diminish the adverse effects of sepsis-induced immunosuppression. © Society for Leukocyte Biology.

  13. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

    Energy Technology Data Exchange (ETDEWEB)

    Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

    1978-01-01

    The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 14/C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 1/ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on ..delta../sup 1/ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.

  14. Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.

    Directory of Open Access Journals (Sweden)

    Philip E Morgan

    Full Text Available Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH. We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC were incubated with high glucose (25 mM, 24 h, 37°C, or methylglyoxal (MGO, glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C, before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05 decreased total thiols (∼35%, further experiments with MGO showed significant losses of GSH (∼40% and NADPH (∼10%; these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10% NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE formed; lower levels of N(ε-(carboxyethyllysine (CEL and N(ε-(carboxymethyllysine (CML were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.

  15. Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

    Science.gov (United States)

    Jančinová, Viera; Pažoureková, Silvia; Lucová, Marianna; Perečko, Tomáš; Mihalová, Danica; Bauerová, Katarína; Nosáľ, Radomír; Drábiková, Katarína

    2015-09-01

    Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCβII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hiroki Moribe

    2012-09-01

    Full Text Available Reactive oxygen species (ROS are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX family enzymes direct the regulated production of ROS. Hydrogen peroxide (H(2O(2 generated by dual oxidases (DUOXs, a member of the NOX family, is crucial for innate mucosal immunity. In addition, H(2O(2 is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA. In the current study, we show that genetic mutation of DUOX (bli-3, DUOXA (doxa-1, and peroxidase (mlt-7 in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H(2O(2 generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H(2O(2. This study provides the first evidence for an essential role of tetraspanin in ROS generation.

  17. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    Science.gov (United States)

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

  18. Differences in Monoamine Oxidase Activity in the Brain of Wistar and August Rats with High and Low Locomotor Activity: A Cytochemical Study.

    Science.gov (United States)

    Sergutina, A V; Rakhmanova, V I

    2016-06-01

    Monoamine oxidase activity was quantitatively assessed by cytochemical method in brain structures (layers III and V of the sensorimotor cortex, caudate nucleus, nucleus accumbens, hippocampal CA3 field) of rats of August line and Wistar population with high and low locomotor activity in the open fi eld test. Monoamine oxidase activity (substrate tryptamine) predominated in the nucleus accumbens of Wistar rats with high motor activity in comparison with rats with low locomotor activity. In August rats, enzyme activity (substrates tryptamine and serotonin) predominated in the hippocampus of animals with high motor activity. Comparison of August rats with low locomotor activity and Wistar rats with high motor activity (i.e. animals demonstrating maximum differences in motor function) revealed significantly higher activity of the enzyme (substrates tryptamine and serotonin) in the hippocampus of Wistar rats. The study demonstrates clear-cut morphochemical specificity of monoaminergic metabolism based on the differences in the cytochemical parameter "monoamine oxidase activity", in the studied brain structures, responsible for the formation and realization of goal-directed behavior in Wistar and August rats.

  19. Association of molecular markers with polyphenol oxidase activity in selected wheat genotypes

    International Nuclear Information System (INIS)

    Abbas, Z.; Javad, B.; Majeed, N.; Naqvi, S.

    2016-01-01

    Wheat (Triticum aestivum L.), a major staple food for the people of Pakistan and other Asian countries, is used as bread, chapatti, porridge, noodles and many other. It is established that color quality of wheat products depend on chemical and enzymatic factors especially the polyphenol oxidases (PPOs). These are copper containing enzymes which induce browning in wheat-based products. Various procedures for determining PPO activity available and differences in PPO activity among wheat genotypes have been documented. In present study, an attempt was made to establish the association of molecular markers with polyphenol oxidase activity in wheat genotypes having very high or very low PPO activities. Twelve pairs of markers were used out of which only three primer pairs viz. PPO43, PPO30 and WP2-2 yielded specific pattern discriminating high and low PPO genotypes. Cluster analysis for all 12 markers revealed that all the low PPO lline share the same sub cluster, but high PPO lines were dispersed in different clusters. (author)

  20. Different responses to heat shock stress revealed heteromorphic adaptation strategy of Pyropia haitanensis (Bangiales, Rhodophyta).

    Science.gov (United States)

    Luo, Qijun; Zhu, Zhenggang; Zhu, Zhujun; Yang, Rui; Qian, Feijian; Chen, Haimin; Yan, Xiaojun

    2014-01-01

    Pyropia has a unique heteromorphic life cycle with alternation stages between thallus and conchocelis, which lives at different water temperatures in different seasons. To better understand the different adaptation strategies for temperature stress, we tried to observe comparative biochemical changes of Pyropia haitanensis based on a short term heat shock model. The results showed that: (1) At normal temperature, free-living conchocelis contains significantly higher levels of H2O2, fatty acid-derived volatiles, the copy number of Phrboh and Phhsp70 genes,the activities of NADPH oxidase and floridoside than those in thallus. The released H2O2 and NADPH oxidase activity of conchocelis were more than 7 times higher than those of thallus. The copy number of Phrboh in conchocelis was 32 times that in thallus. (2) After experiencing heat shock at 35°C for 30 min, the H2O2 contents, the mRNA levels of Phrboh and Phhsp70, NADPH oxidase activity and the floridoside content in thallus were all significantly increased. The mRNA levels of Phrboh increased 5.78 times in 5 min, NADPH oxidase activity increased 8.45 times in 20 min. (3) Whereas, in conchocelis, the changes in fatty acids and their down-stream volatiles predominated, significantly increasing levels of saturated fatty acids and decreasing levels of polyunsaturated fatty acids occurred, and the 8-carbon volatiles were accumulated. However, the changes in H2O2 content and expression of oxidant-related genes and enzymatic activity were not obvious. Overall, these results indicate that conchocelis maintains a high level of active protective apparatus to endure its survival at high temperature, while thallus exhibit typical stress responses to heat shock. It is concluded that Pyropia haitanensis has evolved a delicate strategy for temperature adaptation for its heteromorphic life cycle.

  1. Different responses to heat shock stress revealed heteromorphic adaptation strategy of Pyropia haitanensis (Bangiales, Rhodophyta.

    Directory of Open Access Journals (Sweden)

    Qijun Luo

    Full Text Available Pyropia has a unique heteromorphic life cycle with alternation stages between thallus and conchocelis, which lives at different water temperatures in different seasons. To better understand the different adaptation strategies for temperature stress, we tried to observe comparative biochemical changes of Pyropia haitanensis based on a short term heat shock model. The results showed that: (1 At normal temperature, free-living conchocelis contains significantly higher levels of H2O2, fatty acid-derived volatiles, the copy number of Phrboh and Phhsp70 genes,the activities of NADPH oxidase and floridoside than those in thallus. The released H2O2 and NADPH oxidase activity of conchocelis were more than 7 times higher than those of thallus. The copy number of Phrboh in conchocelis was 32 times that in thallus. (2 After experiencing heat shock at 35°C for 30 min, the H2O2 contents, the mRNA levels of Phrboh and Phhsp70, NADPH oxidase activity and the floridoside content in thallus were all significantly increased. The mRNA levels of Phrboh increased 5.78 times in 5 min, NADPH oxidase activity increased 8.45 times in 20 min. (3 Whereas, in conchocelis, the changes in fatty acids and their down-stream volatiles predominated, significantly increasing levels of saturated fatty acids and decreasing levels of polyunsaturated fatty acids occurred, and the 8-carbon volatiles were accumulated. However, the changes in H2O2 content and expression of oxidant-related genes and enzymatic activity were not obvious. Overall, these results indicate that conchocelis maintains a high level of active protective apparatus to endure its survival at high temperature, while thallus exhibit typical stress responses to heat shock. It is concluded that Pyropia haitanensis has evolved a delicate strategy for temperature adaptation for its heteromorphic life cycle.

  2. Effects of phenylated compounds of methylglyoxal bis(guanylhydrazone) on diamine oxidase activity from rat small intestine.

    Science.gov (United States)

    Balaña-Fouce, R; Pulido, T G; Escudero, D O; Sanz-Sanchez, F

    1986-01-01

    Two phenylated compounds of methylglyoxal bis(guanylhydrazone), potentially inhibitors of diamine oxidase activity, have been synthesized: phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis(guanylhydrazone). Their inhibitory capacity was tested: while PGBG was able to reduce the enzyme activity by 50% at 1.3 microM, DPGBG was only able to reduce diamine oxidase activity by less than 2% at a concentration 1000-fold higher. The inhibition of PGBG was non-competitive and the Ki calculated by a Dixon plot was estimated as 1.7 microM.

  3. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba (Brazil)

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  4. Identification of aldehyde oxidase 1 and aldehyde oxidase homologue 1 as dioxin-inducible genes

    International Nuclear Information System (INIS)

    Rivera, Steven P.; Choi, Hyun Ho; Chapman, Brett; Whitekus, Michael J.; Terao, Mineko; Garattini, Enrico; Hankinson, Oliver

    2005-01-01

    Aldehyde oxidases are a family of highly related molybdo-flavoenzymes acting upon a variety of compounds of industrial and medical importance. We have identified aldehyde oxidase 1 (AOX1) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) inducible gene in the mouse hepatoma cell line Hepa-1. AOX1 mRNA levels were not increased by dioxin in mutant derivatives of the Hepa-1 cell line lacking either functional aryl hydrocarbon receptor (AHR) or aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, thus demonstrating that transcriptional induction of AOX1 in response to dioxin occurs through the AHR pathway. Dioxin induction of AOX1 mRNA was also observed in mouse liver. In addition, levels of AOX1 protein as well as those of aldehyde oxidase homologue 1 (AOH1), a recently identified homolog of AOX1, were elevated in mouse liver in response to dioxin. Employing an aldehyde oxidase specific substrate, AOX1/AOH1 activity was shown to be induced by dioxin in mouse liver. This activity was inhibited by a known inhibitor of aldehyde oxidases, and eliminated by including tungstate in the mouse diet, which is known to lead to inactivation of molybdoflavoenzymes, thus confirming that the enzymatic activity was attributable to AOX1/AOH1. Our observations thus identify two additional xenobiotic metabolizing enzymes induced by dioxin

  5. Metabolic activation of 2-methylfuran by rat microsomal systems

    International Nuclear Information System (INIS)

    Ravindranath, V.; Boyd, M.R.

    1985-01-01

    2-Methylfuran (2-MF), a constituent of cigarette smoke and coffee, causes necrosis of liver, lungs, and kidneys in rodents. 2-MF is metabolically activated by mixed-function oxidases to acetylacrolein, a reactive metabolite that binds covalently to microsomal protein. The hepatic microsomal metabolism of 2-MF to reactive metabolite required the presence of NADPH and oxygen and was dependent on incubation time and substrate concentration. The microsomal metabolism of 2-MF was inducible by pretreatment of rats with phenobarbital and was inhibited by piperonyl butoxide and N-octyl imidazole, which indicates that the metabolism of 2-MF may be mediated by cytochrome P-450. Acetylacrolein was a potent inhibitor of mixed-function oxidase and completely inhibited the microsomal metabolism of 2-MF, indicating that 2-MF is a suicide substrate for the enzyme. The sulfhydryl nucleophile cysteine was a better trapping agent of the reactive metabolite of 2-MF than N-acetylcysteine or glutathione. Lysine decreased the covalent binding of 2-MF metabolites, presumably by reacting with the aldehyde group of acetylacrolein. In addition, in the presence of NADPH, 2-MF was bioactivated by both pulmonary and renal cortical microsomes to reactive metabolites that were covalently bound to microsomal proteins

  6. Isolated sulfite oxidase deficiency.

    Science.gov (United States)

    Rupar, C A; Gillett, J; Gordon, B A; Ramsay, D A; Johnson, J L; Garrett, R M; Rajagopalan, K V; Jung, J H; Bacheyie, G S; Sellers, A R

    1996-12-01

    Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

  7. Metabolic activation of the bladder carcinogen 4-nitrobiphenyl (NBP)

    International Nuclear Information System (INIS)

    Swaminathan, S.

    1986-01-01

    The metabolism of NBP, a dog bladder carcinogen, was examined in vitro using rat liver tissues. NBP was metabolized by enzymes localized both in the microsomes and cytosol. The microsomal enzyme activity was inducible by Aroclor 1254 and phenobarbital. High pressure liquid chromatography analysis of the ethyl acetate extract of the reaction mixture, following incubation of [ 3 H]NBP with NADPH and microsomes, revealed four radioactive and UV absorbing peaks with retention times of 5, 8, 14 and 28 min. The peaks at 8, 14 and 28 min corresponded with 4-aminobiphenyl (ABP), NBP and azoxy biphenyl, respectively. The early eluting component with a retention time of 5 min has been tentatively identified as a ring hydroxylated derivative. In contrast to microsomal metabolism, cytosol-mediated metabolism yielded only one major metabolite identified as ABP. Cytosol-mediate reduction was inhibited by the xanthine oxidase inhibitor allopurinol. In vitro incubation of NBP with NADH and commercial preparations of xanthine oxidase also yielded ABP and the formation of the latter was blocked by allopurinol. Xanthine oxidase catalyzed also the binding of [ 3 H]NBP to DNA and proteins; the binding was inhibited by allopurinol. These data support the hypothesis that the nitro reduction step is involved in the activation of the bladder carcinogen NBP, and that the nitroreductases occur in both the microsomes and cytosol. The cytosolic activity is primarily due to xanthine oxidase

  8. Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

    Science.gov (United States)

    Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

    2005-01-01

    Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

  9. Are colorimetric assays appropriate for measuring phenol oxidase activity in peat soils?

    Science.gov (United States)

    Magdalena M. Wiedermann; Evan S. Kane; Timothy J. Veverica; Erik A. Lilleskov

    2017-01-01

    The activity of extracellular phenol oxidases is believed to play a critical role in decomposition processes in peatlands. The water logged, acidic conditions, and recalcitrant litter from the peatland vegetation, lead to exceptionally high phenolics in the peat. In order to quantify the activity of oxidative enzymes involved in the modification and break down of...

  10. Effect of thimerosal on thyroid hormones metabolism in rats

    Directory of Open Access Journals (Sweden)

    Thiago U Pantaleão

    2017-11-01

    Full Text Available Mercury seems to exert an inhibitory effect on deiodinases, but there are few studies using Thimerosal (TM as the mercury source. We aimed to elucidate the effect of TM on thyroid hormones peripheral metabolism. Adult Wistar female rats received 0.25 μg or 250 μg TM/100 g BW, IM, twice a week, for a month. We evaluated serum total T3 and T4, D1 activity using 125I-rT3 as tracer, and D2 activity using 125I-T4. NADPH oxidase activity was measured by Amplex-red/HRP method and mRNA levels by real time PCR. Serum T4 was increased and T3 decreased by the greatest dose of TM. Even though D1 activity in pituitary and kidney was reduced by the highest dose of TM, hepatic D1 activity and D1 mRNA levels remained unchanged. D2 activity was also significantly decreased by the highest dose of TM in all CNS samples tested, except cerebellum, but D2 mRNA was unaltered. mRNA levels of the tested NADPH oxidases were not affected by TM and NADPH oxidase activity was either unaltered or decreased. Our results indicate that TM might directly interact with deiodinases, inhibiting their activity probably by binding to their selenium catalytic site, without changes in enzyme expression.

  11. Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

    Science.gov (United States)

    Hinkkanen, A; Maly, F E; Decker, K

    1983-04-01

    The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

  12. Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

    Science.gov (United States)

    El-Kemary, Maged; El-Mehasseb, Ibrahim; El-Shamy, Hany

    2018-06-01

    Silver doped cadmium oxide (Ag/CdO) nanoparticles with an average size of 41 nm have been successfully synthesized via thermal decomposition and liquid impregnation technique. The structural characterization has been performed by using several spectroscopic techniques, e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM) and fourier-transform infrared (FT-IR). The catechol oxidase has been studied by UV-visible absorption spectroscopy and fourier-transform infrared as well as the mechanism has been assured by cyclic voltammetry and fluorescence spectroscopy. The results indicate that the oxidation does not occur in the presence of unsupported cadmium oxide particles by silver and in the same time, the catechol oxidase activity of silver doped CdO nanoparticles were improved by about three orders of magnitude than silver ions.

  13. Low platelet monoamine oxidase activity in pathological gambling

    International Nuclear Information System (INIS)

    Carrasco, J.L.; Saiz-Ruiz, J.; Hollander, E.; Cesar, J.; Lopez-Ibor, J.J. Jr.

    1994-01-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.)

  14. Low platelet monoamine oxidase activity in pathological gambling

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco, J.L. [Department of Psychiatry, Centro de Salud Mental, Parla Madrid (Spain); Saiz-Ruiz, J. [Department of Psychiatry and Haematology, Hospital Ramon y Cajal, Madrid (Spain); Hollander, E. [Department of Psychiatry, Mount Sinai School of Medicine, Queens Hospital Center, New York (United States); Cesar, J. [Department of Haematology, Hospital Ramon y Cajal, Madrid (Spain); Lopez-Ibor, J.J. Jr. [Department of Psychiatry, Hospital San Carlos, Complutense University, Madrid (Spain)

    1994-12-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.).

  15. Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

    International Nuclear Information System (INIS)

    Chatfield, J.M.; Armstrong, D.J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N 6 -(Δ 2 -isopentenyl)-adenine-2,8- 3 H (i 6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N 6 -side chain of i 6 Ade

  16. Cadmium induces matrix metalloproteinase-9 expression via ROS-dependent EGFR, NF-kB, and AP-1 pathways in human endothelial cells

    International Nuclear Information System (INIS)

    Lian, Sen; Xia, Yong; Khoi, Pham Ngoc; Ung, Trong Thuan; Yoon, Hyun Joong; Kim, Nam Ho; Kim, Kyung Keun; Jung, Young Do

    2015-01-01

    Highlights: • Cadmium induces MMP-9 expression through NADPH oxidase-derived ROS. • Cadmium induces MMP-9 through EGFR-mediated Akt, Erk1/2 and JNK1/2 signaling pathways. • Akt, MAPKs (Erk1/2 and JNK1/2) functioned as upstream signals of NF-kB and AP-1 respectively, in cadmium-induced MMP-9 in endothelial cells. • ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression in ECV304 cells. - Abstract: Cadmium (Cd), a widespread cumulative pollutant, is a known human carcinogen, associated with inflammation and tumors. Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in tumor metastasis; however, the mechanisms underlying the MMP-9 expression induced by Cd remain obscure in human endothelial cells. Here, Cd elevated MMP-9 expression in dose- and time-dependent manners in human endothelial cells. Cd increased ROS production and the ROS-producing NADPH oxidase. Cd translocates p47 phox , a key subunit of NADPH oxidase, to the cell membrane. Cd also activated the phosphorylation of EGFR, Akt, Erk1/2, and JNK1/2 in addition to promoting NF-kB and AP-1 binding activities. Specific inhibitor and mutagenesis studies showed that EGFR, Akt, Erk1/2, JNK1/2 and transcription factors NF-κB and AP-1 were related to Cd-induced MMP-9 expression in endothelial cells. Akt, Erk1/2, and JNK1/2 functioned as upstream signals in the activation of NF-κB and AP-1, respectively. In addition, N-acetyl-L-cystein (NAC), diphenyleneiodonium chloride (DPI) and apocynin (APO) inhibited the Cd-induced activation of EGFR, Akt, Erk1/2, JNK1/2, and p38 MAPK, indicating that ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression. At present, it states that Cd displayed marked invasiveness in ECV304 cells, which was partially abrogated by MMP-9 neutralizing antibodies. These results demonstrated that Cd induces MMP-9 expression via ROS-dependent EGFR- > Erk1/2, JNK1/2- > AP-1 and EGFR- > Akt- > NF-κB signaling pathways and, in turn

  17. Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Brattsten, L B; Evans, C K; Bonetti, S; Zalkow, L H

    1984-01-01

    Carrot foliage monoterpenes induce cytochrome P-450 up to 2.9-fold, NADPH cytochrome c (P-450) reductase up to 1.6-fold, NADPH-oxidation up to 3.8-fold, aldrin epoxidation up to 1.5-fold in southern armyworm larval midgut tissues when incorporated in their diet at 0.2% for 3 days. Stigmasterol and ergosterol did not substantially induce microsomal oxidase activities and significantly inhibited GSH S-aryltransferase activity and sulfotransferase activity. Coumarin did not substantially affect microsomal oxidase and sulfotransferase activity but is the most potent inducer of GSH S-aryltransferase activity, increasing this activity 7-fold. None of the chemicals is acutely toxic to the sixth instar larvae or affect the larval weight gain except coumarin which significantly depressed the maximal body weight attained.

  18. The C242T polymorphism of the p22-phox gene (CYBA is associated with higher left ventricular mass in Brazilian hypertensive patients

    Directory of Open Access Journals (Sweden)

    Krieger José E

    2011-08-01

    Full Text Available Abstract Background Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA on left ventricular structure in Brazilian hypertensive subjects. Methods We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441 and Vitória (n = 120] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample. Results Genotype frequencies in both samples were consistent with the Hardy- Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height2.7 than those carrying the CC genotype in Campinas (76.8 ± 1.6 vs 70.9 ± 1.4 g/m2.7; p = 0.009, and in Vitória (45.6 ± 1.9 vs 39.9 ± 1.4 g/m2.7; p = 0.023 samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03. Conclusions The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height2.7 and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.

  19. Monoamine oxidase inhibitory activity in tobacco particulate matter: Are harman and norharman the only physiologically relevant inhibitors?

    Science.gov (United States)

    Truman, Penelope; Grounds, Peter; Brennan, Katharine A

    2017-03-01

    Monoamine oxidase inhibition is significant in smokers, but it is still unclear how the inhibition that is seen in the brains and bodies of smokers is brought about. Our aim was to test the contribution of the harman and norharman in tobacco smoke to MAO-A inhibition from tobacco smoke preparations, as part of a re-examination of harman and norharman as the cause of the inhibition of MAO-A inhibition in the brain. Tobacco smoke particulate matter and cigarette smoke particulate matter were prepared and the amounts of harman and norharman measured. The results were compared with the total monoamine oxidase-A inhibitory activity. At a nicotine concentration of 0.6μM (a "physiological" concentration in blood) the total monoamine oxidase-A inhibitory activity measured in these samples was sufficient to inhibit the enzyme by approximately 10%. Of this inhibitory activity, only a small proportion of the total was found to be due to harman and norharman. These results show that harman and norharman provide only a moderate contribution to the total monoamine oxidase-A inhibitory activity of tobacco smoke, perhaps under 10%. This suggests that other inhibitors (either known or unknown) may be more significant contributors to total inhibitory activity than has yet been established, and deserve closer examination. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS.

    Science.gov (United States)

    Nichols, Nicole L; Satriotomo, Irawan; Harrigan, Daniel J; Mitchell, Gordon S

    2015-11-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated super-oxide dismutase-1 (SOD1(G93A)), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1(G93A) (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600 μM; 12 μL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms

  1. ROS mediated selection for increased NADPH availability in Escherichia coli.

    Science.gov (United States)

    Reynolds, Thomas S; Courtney, Colleen M; Erickson, Keesha E; Wolfe, Lisa M; Chatterjee, Anushree; Nagpal, Prashant; Gill, Ryan T

    2017-11-01

    The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli. © 2017 Wiley Periodicals, Inc.

  2. The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

    Science.gov (United States)

    Morrison, Ryan D.; Blobaum, Anna L.; Byers, Frank W.; Santomango, Tammy S.; Bridges, Thomas M.; Stec, Donald; Brewer, Katrina A.; Sanchez-Ponce, Raymundo; Corlew, Melany M.; Rush, Roger; Felts, Andrew S.; Manka, Jason; Bates, Brittney S.; Venable, Daryl F.; Rodriguez, Alice L.; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Emmitte, Kyle A.

    2012-01-01

    Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates. PMID:22711749

  3. SA inhibits complex III activity to generate reactive oxygen species and thereby induces GA overproduction in Ganoderma lucidum

    OpenAIRE

    Rui Liu; Pengfei Cao; Ang Ren; Shengli Wang; Tao Yang; Ting Zhu; Liang Shi; Jing Zhu; Ai-Liang Jiang; Ming-Wen Zhao

    2018-01-01

    Ganoderma lucidum has high commercial value because it produces many active compounds, such as ganoderic acids (GAs). Salicylic acid (SA) was previously reported to induce the biosynthesis of GA in G. lucidum. In this study, we found that SA induces GA biosynthesis by increasing ROS production, and further research found that NADPH oxidase-silenced strains exhibited a partial reduction in the response to SA, resulting in the induction of increased ROS production. Furthermore, the localization...

  4. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    International Nuclear Information System (INIS)

    Kirkensgaard, Kristine G.; Hägglund, Per; Finnie, Christine; Svensson, Birte; Henriksen, Anette

    2009-01-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R cryst of 19.0% and an R free of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation

  5. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Kirkensgaard, Kristine G. [Carlsberg Laboratory (Denmark); Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Hägglund, Per; Finnie, Christine; Svensson, Birte [Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Henriksen, Anette, E-mail: anette@crc.dk [Carlsberg Laboratory (Denmark)

    2009-09-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.

  6. Production of rabbit antibodies against purified Glucose oxidase

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2012-02-01

    Full Text Available Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

  7. Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

    Science.gov (United States)

    Rocha, Sandra M; Saraiva, Tatiana; Cristóvão, Ana C; Ferreira, Raquel; Santos, Tiago; Esteves, Marta; Saraiva, Cláudia; Je, Goun; Cortes, Luísa; Valero, Jorge; Alves, Gilberto; Klibanov, Alexander; Kim, Yoon-Seong; Bernardino, Liliana

    2016-06-04

    Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia

  8. Absorption of enzymatically active 125I-labeled bovine milk xanthine oxidase fed to rabbits

    International Nuclear Information System (INIS)

    Rzucidlo, S.J.; Zikakis, J.P.

    1990-01-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding 125 I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut

  9. Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

    Directory of Open Access Journals (Sweden)

    R.B. Oliveira

    1998-09-01

    Full Text Available The effects of methylmercury (MeHg on histochemical demonstration of the NADPH-diaphorase (NADPH-d activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1 and the other animals were perfused 6 months later (group 2. After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80. Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05. These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

  10. Chronic Granulomatous Disease: The European Experience

    NARCIS (Netherlands)

    van den Berg, J. Merlijn; van Koppen, Elsbeth; Ahlin, Anders; Belohradsky, Bernd H.; Bernatowska, Ewa; Corbeel, Lucien; Español, Teresa; Fischer, Alain; Kurenko-Deptuch, Magdalena; Mouy, Richard; Petropoulou, Theoni; Roesler, Joachim; Seger, Reinhard; Stasia, Marie-José; Valerius, Niels H.; Weening, Ron S.; Wolach, Baruch; Roos, Dirk; Kuijpers, Taco W.

    2009-01-01

    CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst'', essential for the clearance of phagocytosed

  11. Chronic granulomatous disease: the European experience

    NARCIS (Netherlands)

    van den Berg, J.M.; van Koppen, E.; Åhlin, A.; Belohradsky, B.H.; Bernatowska, E.; Corbeel, L.; Español, T.; Fischer, A.; Kurenko-Deptuch, M.; Mouy, R.; Petropoulou, T.; Roesler, J.; Seger, R.; Stasia, M.J.; Valerius, N.H.; Weening, R.S.; Wolach, B.; Roos, D.; Kuijpers, T.W.

    2009-01-01

    CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst'', essential for the clearance of phagocytosed

  12. Regulation of the cellular content of the organic osmolyte taurine in mammalian cells

    DEFF Research Database (Denmark)

    Lambert, Ian H.

    2004-01-01

    regulatory volume decrease, iPLA2, cPLA2, leukotriene D4, reactive oxygen species, NADPH oxidase, protein tyrosine phosphatase, lysophosphatidyl choline......regulatory volume decrease, iPLA2, cPLA2, leukotriene D4, reactive oxygen species, NADPH oxidase, protein tyrosine phosphatase, lysophosphatidyl choline...

  13. Cross talk between MMP2-Spm-Cer-S1P and ERK1/2 in proliferation of pulmonary artery smooth muscle cells under angiotensin II stimulation.

    Science.gov (United States)

    Chowdhury, Animesh; Sarkar, Jaganmay; Pramanik, Pijush Kanti; Chakraborti, Tapati; Chakraborti, Sajal

    2016-08-01

    The aim of the present study is to establish the mechanism associated with the proliferation of PASMCs under ANG II stimulation. The results showed that treatment of PASMCs with ANG II induces an increase in cell proliferation and 100 nM was the optimum concentration for maximum increase in proliferation of the cells. Pretreatment of the cells with AT1, but not AT2, receptor antagonist inhibited ANG II induced cell proliferation. Pretreatment with pharmacological and genetic inhibitors of sphingomyelinase (SMase) and sphingosine kinase (SPHK) prevented ANG II-induced cell proliferation. ANG II has also been shown to induce SMase activity, SPHK phosphorylation and S1P production. In addition, ANG II caused an increase in proMMP-2 expression and activation, ERK1/2 phosphorylation and NADPH oxidase activation. Upon inhibition of MMP-2, SMase activity and S1P level were curbed leading to inhibition of cell proliferation. SPHK was phosphorylated by ERK1/2 during ET-1 stimulation of the cells. ANG II-induced ERK1/2 phosphorylation and proMMP-2 expression and activation in the cells were abrogated upon inhibition of NADPH oxidase activity. Overall, NADPH oxidase plays an important role in proMMP-2 expression and activation and that MMP-2 mediated SMC proliferation occurs through the involvement of Spm-Cer-S1P signaling axis under ANG II stimulation of PASMCs. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

    Science.gov (United States)

    Gudkova, O O; Latyshko, N V; Shandrenko, S G

    2016-01-01

    In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

  15. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    Science.gov (United States)

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects

    DEFF Research Database (Denmark)

    Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads

    2017-01-01

    Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular...... by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2...... %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM-1), endothelial nitric oxide synthase, NADPH oxidase and the Rho...

  17. Role of Nox2 and p22phox in Persistent Postoperative Hypertension in Aldosterone-Producing Adenoma Patients after Adrenalectomy

    Directory of Open Access Journals (Sweden)

    Xiaojing Geng

    2016-01-01

    Full Text Available Adrenal aldosterone-producing adenoma (APA, producing the salt-retaining hormone aldosterone, commonly causes secondary hypertension, which often persists after unilateral adrenalectomy. Although persistent hypertension was correlated with residual hormone aldosterone, the in vivo mechanism remains unclear. NADPH oxidase is the critical cause of aldosterone synthesis in vitro. Nox2 and p22phox comprise the NADPH oxidase catalytic core, serving to initiate a reactive oxygen species (ROS cascade that may participate in the pathology. mRNAs of seven NADPH oxidase isoforms in APA were evaluated by RT-PCR and Q-PCR and their proteins by immunohistochemistry and Western blotting. NADPH oxidase activity was also detected. Nox2 and p22phox were especially abundant in APA. Particularly higher Nox2 and p22phox gene and protein levels were seen in APA than controls. Significant correlations between Nox2 mRNA and aldosterone synthase (CYP11B2 mRNA (R=0.66, P<0.01 and Nox2 protein and baseline plasma aldosterone concentration (PAC (R=0.503, P<0.01 were detected in APA; however, none were found between p22phox mRNA, CYP11B2 mRNA, p22phox protein, and baseline PAC. Importantly, we found that Nox2 localized specifically in hyperplastic zona glomerulosa cells. In conclusion, our results highlight that Nox2 and p22phox may be directly involved in pathological aldosterone production and zona glomerulosa cell proliferation after APA resection.

  18. ABTS assay of phenol oxidase activity in soil.

    Science.gov (United States)

    Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

    2007-12-01

    Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

  19. Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and methyl-salicylate and their relation with calcium signalling.

    Science.gov (United States)

    Garrido, Inmaculada; Espinosa, Francisco; Alvarez-Tinaut, M Carmen

    2009-10-01

    Ca(2+) plays a critical role as second messenger in the signal-response coupling of plant defence responses, and methyl-jasmonate and methyl-salicylate are important components of signal transduction cascades activating plant defences. When intact axenic non-induced seedling roots of sunflower were treated with different Ca(2+) concentrations up to 1 mM, there was no significant increase in O(2)(*-) generation or DMAB-MBTH peroxidase (extracellular, ECPOX) activities in the apoplast, probably because these roots had enough Ca(2+) in their exo- and endocellular reservoirs. Both activities were strongly inhibited by the RBOH-NADPH oxidase inhibitor DPI and by the Ca(2+) surrogate antagonist La(3+), but the voltage-dependent Ca(2+) channel blocker verapamil was only inhibitory at concentrations higher than those active on animal L-type Ca(2+) channels. Concentrations >5 mM EGTA (chelating Ca(2+) in the apoplast) and Li(+) (inhibiting PI cycle dependent endogenous Ca(2+) fluxes) also inhibited both activities. W7, inhibitor of binding of Ca-CaM to its target protein, enhanced both activities, but the inactive analogue W5 showed a similar effect. Our data suggest that Ca(2+) from exocellular and, to a lesser extent, from endocellular stores is involved in oxidative activities, and that RBOH-NADPH oxidase is the main system supporting them. Ca(2+) activation of the PM cytosolic side of RBOH-NADPH oxidase is probably the key to Ca(2+) involvement in these processes. Roots induced by MeJA or MeSA showed significant enhancement of both oxidative activities, as corresponding to the oxidative burst evoked by the two phytohormones in the root apoplast. But while ECPOX activity showed a response to the effectors similar to that described above for non-induced roots, O(2)(*-) generation activity in the apoplast of induced roots was insensitive to EGTA, verapamil and Li(+), the inhibitors of exogenous and endogenous Ca(2+) fluxes; only DPI and La(3+) were inhibitory. As

  20. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    Science.gov (United States)

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-07-09

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

  1. Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

    International Nuclear Information System (INIS)

    Boxtel, Antonius L. van; Kamstra, Jorke H.; Fluitsma, Donna M.; Legler, Juliette

    2010-01-01

    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.

  2. Effect of the dimetilsulfoxido in the response chemiluminescent and the consumption of oxygen of neutrophils activated human

    International Nuclear Information System (INIS)

    Garcia, J.

    2001-01-01

    Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminescent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescense induced by calcium ionophore A23187 was probably due to OH scavenging, whereas inhibition of the lucigenin chemiluminescence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan-induced luminol chemiluminescense was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescense was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescense in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits. These data imply that DMSO inhibits neutrophil chemiluminescense both by OH scavenging and interfering with oxidase activation. Key words:Dimethylsulfoxide, chemiluminescent, luminol, lucigenin,neutrophils [es

  3. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

  4. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  5. Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors.

    Science.gov (United States)

    Hossain, Ekhtear; Anand-Srivastava, Madhu B

    2017-08-01

    We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

  6. Minimizing the cancer-promotional activity of cox-2 as a central strategy in cancer prevention.

    Science.gov (United States)

    McCarty, Mark F

    2012-01-01

    A recent meta-analysis examining long-term mortality in subjects who participated in controlled studies evaluating the impact of daily aspirin on vascular risk, has concluded that aspirin confers substantial protection from cancer mortality. Remarkably, low-dose aspirin was as effective as higher-dose regimens; hence this protection may be achievable with minimal risk. There is reason to believe that this protection stems primarily from inhibition of cox-2 in pre-neoplastic lesions. Since safe aspirin regimens can only achieve a partial and transitory inhibition of cox-2, it may be feasible to complement the cancer-protective benefit of aspirin with other measures which decrease cox-2 expression or which limit the bioactivity of cox-2-derived PGE2. Oxidative stress boosts cox-2 expression by up-regulating activation of NF-kappaB and MAP kinases; NADPH oxidase activation may thus promote carcinogenesis by increasing cox-2 expression while also amplifying oxidant-mediated mutagenesis. A prospective cohort study has observed that relatively elevated serum bilirubin levels are associated with a marked reduction in subsequent cancer mortality; this may reflect bilirubin's physiological role as a potent inhibitor of NADPH oxidase. It may be feasible to mimic this protective effect by supplementing with spirulina, a rich source of a phycobilin which shares bilirubin's ability to inhibit NADPH oxidase. Ancillary antioxidant measures - phase 2 inducing phytochemicals, melatonin, N-acetylcysteine, and astaxanthin - may also aid cox-2 down-regulation. The cancer protection often associated with high-normal vitamin D status may be attributable, in part, to the ability of the activated vitamin D receptor to decrease cox-2 expression while promoting PGE2 catabolism and suppressing the expression of PGE2 receptors. Diets with a relatively low ratio of omega-6 to long-chain omega-3 fats may achieve cancer protection by antagonizing the production and bioactivity of PGE2. Growth

  7. Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Pils, D; Schmetterer, G

    2001-09-25

    Synechocystis sp. PCC 6803 contains three respiratory terminal oxidases (RTOs): cytochrome c oxidase (Cox), quinol oxidase (Cyd), and alternate RTO (ARTO). Mutants lacking combinations of the RTOs were used to characterize these key enzymes of respiration. Pentachlorophenol and 2-heptyl-4-hydroxy-quinoline-N-oxide inhibited Cyd completely, but had little effect on electron transport to the other RTOs. KCN inhibited all three RTOs but the in vivo K(I) for Cox and Cyd was quite different (7 vs. 27 microM), as was their affinity for oxygen (K(M) 1.0 vs. 0.35 microM). ARTO has a very low respiratory activity. However, when uptake of 3-O-methylglucose, an active H+ co-transport, was used to monitor energization of the cytoplasmic membrane, ARTO was similarly effective as the other RTOs. As removal of the gene for cytochrome c(553) had the same effects as removal of ARTO genes, we propose that the ARTO might be a second Cox. The possible functions, localization and regulation of the RTOs are discussed.

  8. Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-a therapy

    NARCIS (Netherlands)

    D. Fekkes (Durk); A.R. van Gool (Arthur); M. Bannink (Marjolein); S. Sleijfer (Stefan); W.H.J. Kruit (Wim); B. van der Holt (Bronno); A.M.M. Eggermont (Alexander); M.W. Hengeveld (Michiel); G. Stoter (Gerrit)

    2009-01-01

    textabstractAbstract Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases

  9. Augmented Rac1 Expression and Activity are Associated with Oxidative Stress and Decline of β Cell Function in Obesity.

    Science.gov (United States)

    Zhou, Shutong; Yu, Dongni; Ning, Shangyong; Zhang, Heli; Jiang, Lei; He, Lei; Li, Miao; Sun, Mingxiao

    2015-01-01

    The aim of this study was to clarify the relationship among Rac1 expression and activation, oxidative stress and β cell dysfunction in obesity. In vivo, serum levels of glucose, insulin, oxidative stress markers and Rac1 expression were compared between ob/ob mice and C57BL/6J controls. Then, these variables were rechecked after the administration of the specific Rac1 inhibitor-NSC23766 in ob/ob mice. In vitro, NIT-1 β cells were cultured in a hyperglycemic and/or hyperlipidemic state with or without NSC23766, and the differences of Rac1 expression and translocation, NADPH oxidase(Nox) enzyme activity, reactive oxygen species (ROS) and insulin mRNA were observed. ob/ob mice displayed abnormal glycometabolism, oxidative stress and excessive expression of Rac1 in the pancreas. NSC23766 injection inhibited the expression of Rac1 in the pancreas, along with amelioration of oxidative stress and glycometabolism in obese mice. Under hyperglycemic and/or hyperlipidemic conditions, Rac1 translocated to the cellular membrane, induced activation of the NADPH oxidase enzyme and oxidative stress, and simultaneously reduced the insulin mRNA expression in NIT-1 β cells. Inhibiting Rac1 activity could alleviate oxidative stress and meliorate the decline of insulin mRNA in β cells. Rac1 might contribute to oxidative stress systemically and locally in the pancreas in obesity. The excessive activation and expression of Rac1 in obesity were associated with β cell dysfunction through ROS production. © 2015 S. Karger AG, Basel.

  10. Effect of temperature stress on polyphenol oxidase activity in grains of some wheat cultivars

    International Nuclear Information System (INIS)

    Kayani, W.K.

    2011-01-01

    Color is a key quality trait of wheat-based products and polyphenol oxidase (PPO) is implicated to play a significant role in their undesirable darkening. Polyphenol oxidase catalyzes the oxidation of phenols to quinines, which auto oxidize and polymerize with amino acid of cellular proteins resulting brown and black pigmentation propounding reduced nutritional values. In present study, the PPO activity in 50 different Pakistani wheat cultivars was investigated and grouped into three categories viz; low, medium and high PPO activity cultivars. PPO is a heat labile enzyme. To investigate effect of heat stress, nine cultivars from each category were chosen for treatment at 30, 40 and 50 deg. C for 30, 60, and 120 minutes each. A substantial change was experienced in PPO activity as compared to room temperature. Two wheat cultivar Wafaq-2001 and AS-2002 showed a compromising attitude of minimum PPO activity at 30 deg. C for a period of 30 and 60 minutes of incubation. In general, an incubation of 30 deg. C or 60 deg. C (low or high) for a period of 30 minutes can be recommended for suppressing PPO activity. (author)

  11. Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Susan J. Gelman

    2018-01-01

    Full Text Available Summary: Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1 occur in multiple types of human cancer. Here, we show that these mutations significantly disrupt NADPH homeostasis by consuming NADPH for 2-hydroxyglutarate (2-HG synthesis. Cells respond to 2-HG synthesis, but not exogenous administration of 2-HG, by increasing pentose phosphate pathway (PPP flux. We show that 2-HG production competes with reductive biosynthesis and the buffering of oxidative stress, processes that also require NADPH. IDH1 mutants have a decreased capacity to synthesize palmitate and an increased sensitivity to oxidative stress. Our results demonstrate that, even when NADPH is limiting, IDH1 mutants continue to synthesize 2-HG at the expense of other NADPH-requiring pathways that are essential for cell viability. Thus, rather than attempting to decrease 2-HG synthesis in the clinic, the consumption of NADPH by mutant IDH1 may be exploited as a metabolic weakness that sensitizes tumor cells to ionizing radiation, a commonly used anti-cancer therapy. : Using liquid chromatography/mass spectrometry (LC/MS and stable isotope tracing, Gelman et al. find that 2-HG production in cells with IDH1 mutations leads to increased pentose phosphate pathway activity to generate NADPH. Production of 2-HG competes with other NADPH-dependent pathways and sensitizes cells to redox stress. Keywords: 2-hydroxyglutarate, cancer metabolism, LC/MS, metabolomcis, pentose phosphate pathway, redox regulation

  12. Mitochondrial type II NAD(PH dehydrogenases in fungal cell death

    Directory of Open Access Journals (Sweden)

    A. Pedro Gonçalves

    2015-03-01

    Full Text Available During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(PH dehydrogenases (also called alternative NAD(PH dehydrogenases are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(PH dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(PH dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF-family.

  13. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    Energy Technology Data Exchange (ETDEWEB)

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A. [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States); Sligar, Stephen G., E-mail: s-sligar@illinois.edu [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States)

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  14. Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

    Science.gov (United States)

    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 4 h 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 4 h LC50 of azadirachtin and allicin, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    Science.gov (United States)

    Jänne, J; Morris, D R

    1984-03-15

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

  16. Structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

    International Nuclear Information System (INIS)

    Yajima, Shunsuke; Hara, Kodai; Iino, Daisuke; Sasaki, Yasuyuki; Kuzuyama, Tomohisa; Ohsawa, Kanju; Seto, Haruo

    2007-01-01

    The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg 2+ was shifted by NADPH in the active site

  17. Gamma radiation affects the anti-Leishmania activity of Bothrops moojeni venom and correlates with L-amino acid oxidase activity

    International Nuclear Information System (INIS)

    Tempone, A.G.; Lourenco, C.O.; Spencer, P.J.; Rogero, J.R.; Nascimento, N.; Andrade Junior, H.F.

    1999-01-01

    Leishmania causes human disfiguring skin disease in endemic areas of Amazon and North Eastern Brazil. Those parasites present a remarkable resistance to most treatments, except those using toxic antimonial salts. We detected a specific anti-Leishmania activity in snake venoms, using an in vitro promastigote assay. In this report, we analyzed the activity of Bothrops moojeni venom against L. Amazonensis, using whole venom or fractions of L-amino acid oxidase (L-AO). Crude venom of B.moojeni, was fractionated by molecular exclusion chromatography. Activity against promastigotes was detected by respiratory oxidative conversion of MTT in a colorimetric assay and L-AO activity was detected by a colorimetric assay with peroxidase and OPD as revealing reagents. Crude venom was irradiated with 500, 1000, and 2000 Gy in a 60 Co gamma radiation source. The venom had an anti-Leishmania activity of 33 pg/promastigote and the active fraction migrates as 100-150 kDa, close to the size described for L-AOs, and also presented L-AO activity. The radiation reduces both the L-AO and anti-Leishmania activity in a dose dependent effect. Those data suggests the anti-Leishmania activity in this venom is closely related to the L-amino acid oxidase activity and also that radiation could be used as a tool to detect specific activities reduction in water solutions, similarly to observed in dry preparations. (author)

  18. Effects of dark chocolate on NOX-2-generated oxidative stress in patients with non-alcoholic steatohepatitis.

    Science.gov (United States)

    Loffredo, L; Del Ben, M; Perri, L; Carnevale, R; Nocella, C; Catasca, E; Baratta, F; Ceci, F; Polimeni, L; Gozzo, P; Violi, F; Angelico, F

    2016-08-01

    Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is considered a pathogenetic mechanism determining fibrosis and disease progression in non-alcoholic steatohepatitis (NASH). Polyphenols exert antioxidant action and inhibit NADPH oxidase in humans. To analyse the effect of cocoa polyphenols on NADPH oxidase isoform 2 (NOX2) activation, oxidative stress and hepatocyte apoptosis in a population affected by NASH. In a cross-sectional study comparing 19 NASH and 19 controls, oxidative stress, as assessed by serum NOX2 activity and F2-isoprostanes, and hepatocyte apoptosis, as assessed by serum cytokeratin-18 (CK-18) levels, were measured. Furthermore, the 19 NASH patients were randomly allocated in a crossover design to 40 g/day of dark chocolate (>85% cocoa) or 40 g/day of milk chocolate (chocolate intake. Compared to controls, NASH patients had higher sNOX2-dp, serum isoprostanes and CK-18 levels. A significant difference for treatments was found in subjects with respect to sNOX2-dp, serum isoprostanes and serum CK-18. The pairwise comparisons showed that, compared to baseline, after 14 days of dark chocolate intake, a significant reduction in sNOX2-dp serum isoprostanes and CK-18 M30 was found. No change was observed after milk chocolate ingestion. A simple linear regression analysis showed that ∆ of sNOX2-dp was associated with ∆ of serum isoprostanes. Cocoa polyphenols exert an antioxidant activity via NOX2 down-regulation in NASH patients. © 2016 John Wiley & Sons Ltd.

  19. High glucose impairs superoxide production from isolated blood neutrophils

    DEFF Research Database (Denmark)

    Perner, A; Nielsen, S E; Rask-Madsen, J

    2003-01-01

    Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This study...... measured the acute effects of high glucose or the G6PD inhibitor dehydroepiandrosterone (DHEA) on the production of O(2)(-) from isolated human neutrophils....

  20. Production of rabbit antibodies against purified Glucose oxidase

    OpenAIRE

    Zia,Muhammad Anjum; Ain,Qurat-ul; Iftikhar,Tehreema; Abbas,Rao Zahid; Rahman,Khalil-ur

    2012-01-01

    Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex ...

  1. Dextromethorphan inhibition of voltage-gated proton currents in BV2 microglial cells.

    Science.gov (United States)

    Song, Jin-Ho; Yeh, Jay Z

    2012-05-10

    Dextromethorphan, an antitussive drug, has a neuroprotective property as evidenced by its inhibition of microglial production of pro-inflammatory cytokines and reactive oxygen species. The microglial activation requires NADPH oxidase activity, which is sustained by voltage-gated proton channels in microglia as they dissipate an intracellular acid buildup. In the present study, we examined the effect of dextromethorphan on proton currents in microglial BV2 cells. Dextromethorphan reversibly inhibited proton currents with an IC(50) value of 51.7 μM at an intracellular/extracellular pH gradient of 5.5/7.3. Dextromethorphan did not change the reversal potential or the voltage dependence of the gating. Dextrorphan and 3-hydroxymorphinan, major metabolites of dextromethorphan, and dextromethorphan methiodide were ineffective in inhibiting proton currents. The results indicate that dextromethorphan inhibition of proton currents would suppress NADPH oxidase activity and, eventually, microglial activation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Magnetic field effects on brain monoamine oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Borets, V.M.; Ostrovskiy, V.Yu.; Bankovskiy, A.A.; Dudinskaya, T.F.

    1985-03-01

    In view of the increasing use of magnetotherapy, studies were conducted on the effects of 35 mTesla magnetic fields on monoamine oxidase activity in the rat brain. Under in vitro conditions a constant magnetic field in the continuous mode was most effective in inhibiting deamination of dopamine following 1 min exposure, while in vivo studies with 8 min or 10 day exposures showed that inhibition was obtained only with a variable field in the continuous mode. However, inhibition of dopamine deamination was only evident within the first 24 h after exposure was terminated. In addition, in none of the cases was norepinephrine deamination inhibited. The effects of the magnetic fields were, therefore, transient and selective with the CNS as the target system. 9 references.

  3. Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats.

    Science.gov (United States)

    Li, Jiajia; Zhang, Rong; Wang, Caixia; Wang, Xin; Xu, Man; Ma, Jingxue; Shang, Qingli

    2018-03-30

    Choroidal neovascularization (CNV) is a common vision-threatening complication associated with many  fundus diseases. The retinal pigment epithelial (RPE) cell junction barrier has critical functions in preventing CNV, and oxidative stress can cause compromise of barrier integrity and induce angiogenesis. Rap1, a small guanosine triphosphatase (GTPase), is involved in regulating endothelial and epithelial cell junctions. In this work, we explored the function and mechanism of Rap1 in CNV in vivo. A laser-induced rat CNV model was developed. Rap1 was activated through intravitreal injection of the Rap1 activator 8CPT-2'-O-Me-cAMP (8CPT). At 14 days after laser treatment, CNV size in RPE/choroid flat mounts was measured by fluorescein isothiocyanate-dextran staining. Expression of vascular endothelial growth factor (VEGF) and cell junction proteins in RPE/choroid tissues were analyzed by western blots and quantitative real-time PCR assays. Reactive oxygen species (ROS) in RPE cells were detectedbydichloro-dihydro-fluorescein diacetate assays. The antioxidant apocynin was intraperitoneally injected into rats. Activating Rap1 by 8CPT significantly reduced CNV size and VEGF expression in the rat CNV model. Rap1 activation enhanced protein and mRNA levels of ZO-1 and occludin, two tight junction proteins in the RPE barrier. In addition, reducing ROS generation by injection of apocynin, a NADPH oxidase inhibitor, inhibited CNV formation. Rap1 activation reduced ROS generation and expression of NADPH oxidase 4. Rap1 activation inhibits CNV through regulating barrier integrity and ROS generation of RPE in vivo, and selectively activating Rap1 may be a way to reduce vision loss from CNV.

  4. Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite.

    Science.gov (United States)

    Pifferi, P G; Vaccari, A; Ricci, G; Poli, G; Ruggeri, O

    1982-10-01

    The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.

  5. NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

    Science.gov (United States)

    Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

    2018-01-20

    The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

  6. Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

    Science.gov (United States)

    Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

    2018-04-03

    While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

  7. Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

    Science.gov (United States)

    Semprun-Prieto, Laura C.; Sukhanov, Sergiy; Yoshida, Tadashi; Rezk, Bashir M.; Gonzalez-Villalobos, Romer A.; Vaughn, Charlotte; Tabony, A. Michael; Delafontaine, Patrice

    2011-01-01

    Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4 fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS. PMID:21570954

  8. [Effect of Kaixinsan on monoamine oxidase activity].

    Science.gov (United States)

    Wang, Shi; Dong, Xian-Zhe; Tan, Xiao; Wang, Yu-Ning; Liu, Ping

    2016-05-01

    To observe the effect of antidepressant medicine prescription, Kaixinsan (KXS) on monoamine oxidase (MAO) activity, and explore the mechanism of KXS in elevating the levels of monoamine neurotransmitter from the perspective of metabolism, in vitro enzyme reaction system and C6 neuroglial cells, the effect of KXS at different concentrations on MAO-A and MAO-B activity was observed. In animal studies, the effect of KXS at different concentrations on MAO-A and MAO-B activities of brain mitochondrialin normal rats and solitary chronic unpredictable moderate stress (CMS) model rats after intragastric administration for 1, 2, 3 weeks. Results showed that 10 g•L⁻¹ KXS could significantly reduce the activity of MAO-A and MAO-B in enzyme reaction system; and in C6 cells, KXS within 0.625-10 g•L⁻¹ concentration range had no significant effect on the activity of MAO-A, but had obvious inhibitory effect on the activity of MAO-B in a dose dependent manner. KXS had no significant effect on the activity of MAO-A and MAO-B in brains of normal rats after action for 1, 2, 3 weeks. After 2 and 3 weeks treatment with 338 mg•kg⁻¹ dose KXS, MAO-A activity in the brain of CMS rats was decreased as compared with the model group (PMAO-B activity after 1, 2, 3 weeks of treatment. The results indicated that KXS had certain effect on in vitro MAO-A and MAO-B activity, had no effect on brain MAO-A and MAO-B activity in vivo in normal rats, and had certain inhibitory effect on MAO-A activity in brains of CMS rats. Copyright© by the Chinese Pharmaceutical Association.

  9. Optimization of Glucose oxidase towards oxygen independency and high mediator activity for amperometric glucose determination in diabetes analytics

    OpenAIRE

    Arango Gutierrez, Erik Uwe

    2015-01-01

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies, and one simultaneous site saturation library (OmniC...

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

    NARCIS (Netherlands)

    Orru, Roberto; Torres Pazmino, Daniel; 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)

  11. Diagnosis and therapy of dysfunctions of human leukocytes after irradiation. Final report; Diagnose und Therapie von Funktionsstoerungen menschlicher Leukozyten nach Bestrahlung. Abschlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Beuningen, D. van; Kaffenberger, W. [Sanitaetsakademie der Bundeswehr, Muenchen (Germany); Baaske, C.; Leipert, D.

    1999-08-01

    Experiments were performed with isolated human PMN and with the promyelocytic HL-60 cell line, induced to differentiate along the granulocytic lineage with dimethyl sulfoxid. The respiratory burst reaction was triggered with soluble stimuli (chemotactic agent: formylated tripeptide, f-MLP, immune complexes, or phorbol ester) and measured flow cytometrically or as chemiluminescence signals with indicator molecules dihydrorhodamine 123 or luminol, respectively. The presence of enzymes was postulated, verified with Western blotting, and their activities were inhibited pharmacologically, lipid 2{sup nd}-messengers (diacylglycerol) were measured by HPLC. Our results identify protein kinase (PK) C activity as the central element of signal transduction cascades involved in the activation of the NADPH oxidase. In addition, several phospholipases ({beta}, {gamma}), protein tyrosine- as well as protein serine/threonine kinases and DAG kinases in combination with phosphatidate phosphohydrolase contribute to intracellular signal transduction processes. For the first time, besides PKC activity new cytoplasmic/membrane-bound elements of signal transduction processes (DAG concentration and DAG kinase activity, phosphatidylinositol 3-kinase activity), and the FC{gamma} receptor-mediated respiratory burst of PMN were identified as radiosensitive ''targets'' for doses < 5 Gy. Theses results provide a basis for causally-oriented therapeutic approaches and could help to explain immunodeficiencies observed after exposure to ionizing radiation. (orig./MG) [German] Die intrazellulaeren Signaluebertragungsprozesse zur Aktivierung der NADPH-Oxidase sind Gegenstand der vorliegenden Untersuchung an bestrahlten PMN des Menschen und an einem PMN-Modell: HL-60 Promyelozyten, die mit Dimethylsulfoxid zur Differenzierung zu Neutrophilen-aehnlichen induziert wurden. Behandlung der Zellen mit loeslichen Rezeptor-abhaengigen oder -unabhaengigen Stimuli (Chemotaxin und

  12. Gene Expression Patterns during Light and Dark Infection of Prochlorococcus by Cyanophage.

    Directory of Open Access Journals (Sweden)

    Luke R Thompson

    Full Text Available Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP, thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2 and its host (Prochlorococcus MED4 throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase-implicated in protecting the photosynthetic membrane from light stress-were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin-NADP+ reductase (FNR, which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under

  13. Gamma radiation affects the anti-Leishmania activity of Bothrops moojeni venom and correlates with L-amino acid oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Tempone, A.G.; Lourenco, C.O.; Spencer, P.J.; Rogero, J.R.; Nascimento, N. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Div. de Radiobiologia; Andrade Junior, H.F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical

    1999-11-01

    Leishmania causes human disfiguring skin disease in endemic areas of Amazon and North Eastern Brazil. Those parasites present a remarkable resistance to most treatments, except those using toxic antimonial salts. We detected a specific anti-Leishmania activity in snake venoms, using an in vitro promastigote assay. In this report, we analyzed the activity of Bothrops moojeni venom against L. Amazonensis, using whole venom or fractions of L-amino acid oxidase (L-AO). Crude venom of B.moojeni, was fractionated by molecular exclusion chromatography. Activity against promastigotes was detected by respiratory oxidative conversion of MTT in a colorimetric assay and L-AO activity was detected by a colorimetric assay with peroxidase and OPD as revealing reagents. Crude venom was irradiated with 500, 1000, and 2000 Gy in a {sup 60} Co gamma radiation source. The venom had an anti-Leishmania activity of 33 pg/promastigote and the active fraction migrates as 100-150 kDa, close to the size described for L-AOs, and also presented L-AO activity. The radiation reduces both the L-AO and anti-Leishmania activity in a dose dependent effect. Those data suggests the anti-Leishmania activity in this venom is closely related to the L-amino acid oxidase activity and also that radiation could be used as a tool to detect specific activities reduction in water solutions, similarly to observed in dry preparations. (author) 13 refs., 3 figs.

  14. Hierarchical CNFs/MnCo2O4.5 nanofibers as a highly active oxidase mimetic and its application in biosensing

    Science.gov (United States)

    Gao, Mu; Lu, Xiaofeng; Nie, Guangdi; Chi, Maoqiang; Wang, Ce

    2017-12-01

    Recently, much attention has been paid on the nanomaterial-based artificial enzymes due to their tunable catalytic activity, high stability and low cost compared to the natural enzymes. Different from the peroxidase mimics which have been studied for several decades, nanomaterials with oxidase-like property are burgeoning in the recent years. In this paper, hierarchical carbon nanofibers (CNFs)/MnCo2O4.5 nanofibers as efficient oxidase mimics are reported. The products are synthesized by an electrospinning technique and an electrochemcial deposition process in which the CNFs are used as the working electrode where MnCo2O4.5 nanosheets deposit on. The resulting binary metal oxide-based nanocomposites exhibit a good oxidase-like activity toward the oxidations of 3,3‧,5,5‧tetramethylbenzi-dine (TMB), 2,2‧-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) salt and o-phenylenediamine (OPD) without exogenous addition of H2O2. The system of CNFs/MnCo2O4.5-TMB can be used as a candidate to detect sulfite and ascorbic acid via a colorimetric method with a high sensitivity. This work provides the efficient utilization and potential applications of binary metal oxide-based nanocomposites with oxidase activities in biosensors and other biotechnologies.

  15. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A2

    International Nuclear Information System (INIS)

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-01-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1- 14 C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A 2 (PLA 2 ) activity (64.3-545.6 nmols/min/mg). The PLA 2 was maximally active in the neutral-alkaline pH range, was Ca 2+ -dependent, and was unaffected by the addition of xanthine. PLA 2 activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA 2 activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca 2+ -dependent PLA 2 measured in various tissue homogenates (≤ 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA 2 may have influenced previously published reports, and such studies should be interpreted cautiously

  16. Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Kučera, J.; Binó, Lucia; Štefková, K.; Jaros, J.; Vašíček, Ondřej; Veceřa, J.; Kubala, Lukáš; Pachernik, J.

    2016-01-01

    Roč. 2016, č.2016 (2016), č. článku 7409196. ISSN 1942-0900 Institutional support: RVO:68081707 Keywords : nadph oxidase inhibitor * smooth-muscle-cells * wnt-beta-catenin Subject RIV: BO - Biophysics Impact factor: 4.593, year: 2016

  17. Ultra-fast HPM detectors improve NAD(P)H FLIM

    Science.gov (United States)

    Becker, Wolfgang; Wetzker, Cornelia; Benda, Aleš

    2018-02-01

    Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.

  18. Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

    Science.gov (United States)

    Wu, Qian; Zhong, Zhao-Ming; Zhu, Si-Yuan; Liao, Cong-Rui; Pan, Ying; Zeng, Ji-Huan; Zheng, Shuai; Ding, Ruo-Ting; Lin, Qing-Song; Ye, Qing; Ye, Wen-Bin; Li, Wei; Chen, Jian-Ting

    2016-01-01

    Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

  19. Sesamin Ameliorates Advanced Glycation End Products-Induced Pancreatic β-Cell Dysfunction and Apoptosis

    Directory of Open Access Journals (Sweden)

    Xiang Kong

    2015-06-01

    Full Text Available Advanced glycation end products (AGEs, the direct modulators of β-cells, have been shown to cause insulin-producing β-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked β-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg and orally treated with sesamin (160 mg/kg for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and β-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 μM and then exposed to AGEs (200 mg/L for 24 h. Insulin secretion, β-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced β-cell dysfunction and apoptosis both in vivo and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67phox and p22phox, and reduced NADPH oxidase activity. These results suggest that sesamin protects β-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.

  20. Hyperoxia-induced p47phox activation and ROS generation is mediated through S1P transporter Spns2, and S1P/S1P1&2 signaling axis in lung endothelium.

    Science.gov (United States)

    Harijith, Anantha; Pendyala, Srikanth; Ebenezer, David L; Ha, Alison W; Fu, Panfeng; Wang, Yue-Ting; Ma, Ke; Toth, Peter T; Berdyshev, Evgeny V; Kanteti, Prasad; Natarajan, Viswanathan

    2016-08-01

    Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS)-induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase. Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia-mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1(-/-) mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1(+/-)) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1&2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47(phox) translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia-mediated ROS generation. In addition, p47(phox) (phox:phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47(phox) and production of ROS involved in hyperoxia-mediated lung injury in neonatal and adult mice. Copyright © 2016 the American Physiological Society.

  1. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    Science.gov (United States)

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  2. Hypoxia sensing in the fetal chicken femoral artery is mediated by the mitochondrial electron transport chain

    DEFF Research Database (Denmark)

    Zoer, Bea; Cogolludo, Angel L; Perez-Vizcaino, Francisco

    2010-01-01

    ) (polyethylene glycol-catalase) or by NADPH-oxidase inhibition (apocynin). Also, the K(+) channel inhibitors tetraethylammonium (nonselective), diphenyl phosphine oxide-1 (voltage-gated K(+) channel 1.5), glibenclamide (ATP-sensitive K(+) channel), iberiotoxin (large-conductance Ca(2+)-activated K(+) channel...

  3. Skewing of X-chromosome inactivation in three generations of carriers with X-linked chronic granulomatous disease within one family

    NARCIS (Netherlands)

    Köker, M. Y.; Sanal, O.; de Boer, M.; Tezcan, I.; Metin, A.; Tan, C.; Ersoy, F.; Roos, D.

    2006-01-01

    BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by impairment of intracellular microbicidal activity of phagocytes. Mutations in one of the four known NADPH-oxidase components preclude generation of superoxide and related

  4. Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

    Directory of Open Access Journals (Sweden)

    Katharina Hahn

    2017-12-01

    Full Text Available l-Amino acid oxidases (l-AAO catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

  5. Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications

    Directory of Open Access Journals (Sweden)

    Arthur I. Cederbaum

    2015-04-01

    Full Text Available The cytochrome P450 mixed function oxidase enzymes play a major role in the metabolism of important endogenous substrates as well as in the biotransformation of xenobiotics. The liver P450 system is the most active in metabolism of exogenous substrates. This review briefly describes the liver P450 (CYP mixed function oxidase system with respect to its enzymatic components and functions. Electron transfer by the NADPH-P450 oxidoreductase is required for reduction of the heme of P450, necessary for binding of molecular oxygen. Binding of substrates to P450 produce substrate binding spectra. The P450 catalytic cycle is complex and rate-limiting steps are not clear. Many types of chemical reactions can be catalyzed by P450 enzymes, making this family among the most diverse catalysts known. There are multiple forms of P450s arranged into families based on structural homology. The major drug metabolizing CYPs are discussed with respect to typical substrates, inducers and inhibitors and their polymorphic forms. The composition of CYPs in humans varies considerably among individuals because of sex and age differences, the influence of diet, liver disease, presence of potential inducers and/or inhibitors. Because of such factors and CYP polymorphisms, and overlapping drug specificity, there is a large variability in the content and composition of P450 enzymes among individuals. This can result in large variations in drug metabolism by humans and often can contribute to drug–drug interactions and adverse drug reactions. Because of many of the above factors, especially CYP polymorphisms, there has been much interest in personalized medicine especially with respect to which CYPs and which of their polymorphic forms are present in order to attempt to determine what drug therapy and what dosage would reflect the best therapeutic strategy in treating individual patients.

  6. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

    Science.gov (United States)

    Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

    2017-01-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

  7. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

    Science.gov (United States)

    Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

    2017-06-01

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Myeloid-specific deletion of NOX2 prevents the metabolic and neurologic consequences of high fat diet.

    Directory of Open Access Journals (Sweden)

    Jennifer K Pepping

    Full Text Available High fat diet-induced obesity is associated with inflammatory and oxidative signaling in macrophages that likely participates in metabolic and physiologic impairment. One key factor that could drive pathologic changes in macrophages is the pro-inflammatory, pro-oxidant enzyme NADPH oxidase. However, NADPH oxidase is a pleiotropic enzyme with both pathologic and physiologic functions, ruling out indiscriminant NADPH oxidase inhibition as a viable therapy. To determine if targeted inhibition of monocyte/macrophage NADPH oxidase could mitigate obesity pathology, we generated mice that lack the NADPH oxidase catalytic subunit NOX2 in myeloid lineage cells. C57Bl/6 control (NOX2-FL and myeloid-deficient NOX2 (mNOX2-KO mice were given high fat diet for 16 weeks, and subject to comprehensive metabolic, behavioral, and biochemical analyses. Data show that mNOX2-KO mice had lower body weight, delayed adiposity, attenuated visceral inflammation, and decreased macrophage infiltration and cell injury in visceral adipose relative to control NOX2-FL mice. Moreover, the effects of high fat diet on glucose regulation and circulating lipids were attenuated in mNOX2-KO mice. Finally, memory was impaired and markers of brain injury increased in NOX2-FL, but not mNOX2-KO mice. Collectively, these data indicate that NOX2 signaling in macrophages participates in the pathogenesis of obesity, and reinforce a key role for macrophage inflammation in diet-induced metabolic and neurologic decline. Development of macrophage/immune-specific NOX-based therapies could thus potentially be used to preserve metabolic and neurologic function in the context of obesity.

  9. Sex-related differences in NADPH-dependent lipid peroxidation induced by cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Masao; Nagai, Yasushi

    1986-10-01

    Male and female rats were dosed once a day for 2 days with injections of 1.5 mg Cd/kg. Formation of thiobarbituric acid reactive substances (TBA-RS) was significantly increased in male rat liver but not in the females. NADPH-dependent lipid peroxidation in vitro in microsomes derived from untreated rat liver was greater in males than in females. Furthermore, addition of cadmium (Cd) to microsomes isolated from male rat liver produced a dose-dependent potentiation of NADPH-dependent lipid peroxidation from low concentrations of CD. In microsomes derived from females a significant increase in lipid peroxidation was observed only at high Cd concentrations. NADPH-dependent lipid peroxidation enhanced by Cd was greater in the males than in the females. These data suggest that a sex-related difference in the ability of Cd to induce lipid peroxidation in vivo in rat liver appears to be mediated partly through differences in hepatic microsomal NADPH-dependent lipid peroxidation.

  10. Structure-Activity Relationship Analysis of 3-phenylcoumarin-Based Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Rauhamäki, Sanna; Postila, Pekka A.; Niinivehmas, Sanna; Kortet, Sami; Schildt, Emmi; Pasanen, Mira; Manivannan, Elangovan; Ahinko, Mira; Koskimies, Pasi; Nyberg, Niina; Huuskonen, Pasi; Multamäki, Elina; Pasanen, Markku; Juvonen, Risto O.; Raunio, Hannu; Huuskonen, Juhani; Pentikäinen, Olli T.

    2018-03-01

    Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson’s disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM - 1 µM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.

  11. Change of the NADPH depending superoxide producing and ferri hemoglobin reducing activities of cytochrome b558 from spleen cells and erythrocytes membranes induced by the radiation of different character

    International Nuclear Information System (INIS)

    Melkonyan, L.G.; Simonyan, R.M.; Simonyan, M.A.; Sekoyan, E.S.

    2009-01-01

    After the X radiation, UVA radiation and ultrasound radiation of new isoforms of cytochrome cyt b 5 58 from rats erythrocyte membranes - EM (cyt b 5 58III) and from spleen cell membranes (SCM) in vitro, as well as after the radiation of EM ex vivo, the suppression of both NADPH depending O 2 - producing and ferrihemoglobin (ferriHb)-reducing activities of cyt b 5 58 from EM and SCM in homogeneous (in solution) and heterogeneous phases (in EM and SCM) at various scopes takes place. These changes are associated with the destabilization of EM and SCM, conditioned by the change of the aggregation degree of these hemoproteins in EM and SCM, hemoproteins as a result of the influence of the hydrogen peroxide formed during radiolysis and photolysis of the water medium. After He-Ne laser radiation of the cyt b 5 58 from EM and SCM in vitro an increase of the NADPH depending O 2 - producing and ferriHb-reducing activities of the cyt b 5 58 from EM and SCM in homogenous and heterogeneous phases (in membranes) takes place. It is supposed that the suppression (by X-, UVA- and US-radiation) and the stimulation (by He-Ne laser radiation) of the immune system activity and the oxygen homeostasis are associated with the corresponding decrease and increase of the NADPH depending O 2 - producing and ferriHb-reducings activity of the new isoforms of cyt b 5 58 from EM and SCM in homogeneous and heterogeneous phases

  12. Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

    Directory of Open Access Journals (Sweden)

    Schudt Christian

    2005-07-01

    Full Text Available Abstract Background The sources and measurement of reactive oxygen species (ROS in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR with spin trapping is a specific method for ROS detection, and may address some these technical problems. Methods We have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe l-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway. Results While perfusing lungs with CPH over a range of inspired oxygen concentrations (1–21 %, the rate of CP• formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA into the pulmonary artery caused a rapid increase in CP• formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5 % O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases. Conclusion The perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to

  13. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

    1989-09-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

  14. Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

    International Nuclear Information System (INIS)

    Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

    1989-01-01

    The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31 P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well

  15. NoxO1 Controls Proliferation of Colon Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Franziska Moll

    2018-05-01

    Full Text Available AimReactive oxygen species (ROS produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.ResultsNoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.ConclusionNoxO1 affects colon epithelium homeostasis and prevents inflammation.

  16. Immobilization of xanthine oxidase on a polyaniline silicone support.

    Science.gov (United States)

    Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

    1996-03-01

    A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

  17. Assays of D-Amino Acid Oxidase Activity

    Directory of Open Access Journals (Sweden)

    Elena Rosini

    2018-01-01

    Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

  18. Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK.

    Science.gov (United States)

    Martínez-Revelles, Sonia; García-Redondo, Ana B; Avendaño, María S; Varona, Saray; Palao, Teresa; Orriols, Mar; Roque, Fernanda R; Fortuño, Ana; Touyz, Rhian M; Martínez-González, Jose; Salaices, Mercedes; Rodríguez, Cristina; Briones, Ana M

    2017-09-01

    Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H 2 O 2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with β-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H 2 O 2 and O 2 .- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H 2 O 2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27

  19. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    Science.gov (United States)

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  20. Cellular antioxidant effects of atorvastatin in vitro and in vivo.

    Science.gov (United States)

    Wassmann, Sven; Laufs, Ulrich; Müller, Kirsten; Konkol, Christian; Ahlbory, Katja; Bäumer, Anselm T; Linz, Wolfgang; Böhm, Michael; Nickenig, Georg

    2002-02-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat

  1. The role of free radicals and stress signalling in persistent genomic instability induced by long wavelength UV light

    International Nuclear Information System (INIS)

    Phillipson, R.; McMillan, T.J.

    2003-01-01

    Induction of persistent genomic instability has commonly been investigated with ionising radiation. It has been characterised as a decrease in plating efficiency, and an increase in chromosomal aberrations and mutation frequency in the progeny of cells that survive the initial irradiation. We now present data demonstrating the phenomenon following exposure to long-wavelength solar UV-A (320-400nm) radiation at environmentally relevant doses. Using the spontaneously immortalised human skin keratinocyte line, HaCaT, we observed a significant decrease in plating efficiency (77 +/- 2% of control), and increase in micronuclei (2.5 fold) and mutation frequency (2 fold), 7 days after the initial radiation insult. Modification of UV-A-induced instability by incubation with exogenous catalase implicated reactive oxygen species (ROS), in-particular hydrogen peroxide, in the production and/or maintenance of the phenomenon. Assessment of anti-oxidant enzymes revealed a significant increase in glutathione-s-transferase activity (158 +/- 4% of control) at day 7 in the irradiated cell population, which was inhibited by incubation with exogenous catalase (97 +/- 3%), providing further evidence for an ROS-mediated pathway. Furthermore, inhibition of UV-A-induced micronuclei at day 7 by the flavonoid-containing-protein inhibitor diphenyleneiodonium (DPI) indicates that the NADPH oxidase family of enzymes may be involved in this phenomenon. Measurement of superoxide production by the cytochrome c reduction assay revealed that the irradiated cell population produce 50% more superoxide than the unirradiated controls, and that incubation with DPI led to a preferential reduction in superoxide production in the UV-A treated population at day 7. Finally, NADPH oxidase activity is increased significantly over controls in UV-A-treated cells. These data demonstrate that oxidative stress, analogous to that produced by ionising radiation, induces persistent genomic instability through a

  2. Pancreatic Beta-Cell Purification by Altering FAD and NAD(PH Metabolism

    Directory of Open Access Journals (Sweden)

    P. de Vos

    2008-07-01

    Full Text Available Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD and nicotinamide-adenine dinucleotide phosphate (NAD(PH autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(PH fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(PH contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(PH fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

  3. Modulating effect of new potential antimelanomic agents, spin-labeled triazenes and nitrosoureas on the DOPA-oxidase activity of tyrosinase.

    Science.gov (United States)

    Gadjeva, V; Zheleva, A; Raikova, E

    1999-07-01

    The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity.

  4. Preliminary pharmacological studies on Eugenia uniflora leaves: xanthine oxidase inhibitory activity.

    Science.gov (United States)

    Schmeda-Hirschmann, G; Theoduloz, C; Franco, L; Ferro, E; de Arias, A R

    1987-11-01

    Eugenia uniflora is widely used in Paraguayan folk medicine. A hydroalcoholic extract of the leaves showed some central nervous system activity in hippocratic screening when given intraperitoneally, but little to no acute or subacute toxicity in doses up to 4200 mg/kg orally in BALB c mice. The LD50 of the extract was 220 mg/kg i.p. in mice. A decoction or infusion of the leaves is recommended for treating gout by native herbalists. The known flavonoids quercitrin, quercetin, myricitrin and myricetin were found to be responsible for the xanthine oxidase inhibitory action of the plant extract.

  5. NADPH-diaphorase expression in the Meibomian glands of rat palpebra in postnatal development

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    D. Kluchova

    2010-11-01

    Full Text Available In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS positive nerve fibers in rat meibomian glands (MGs at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d is a surrogate for neuronal nitric oxide synthase (NOS. Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.

  6. Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

    International Nuclear Information System (INIS)

    Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

    2011-01-01

    We had earlier shown that exposure to arsenic (0.50 μM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca 2+ ) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca 2+ homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca 2+ levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: → Altered Ca 2+ homeostasis leads to arsenic-induced HKM apoptosis. → Calpain-2 plays a critical role in the process. → ERK is pro-apoptotic in arsenic-induced HKM apoptosis. → Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

  7. A study of monoamine oxidase activity in fetal membranes.

    Science.gov (United States)

    Sekizawa, A; Ishikawa, H; Morimoto, T; Hirose, K; Suzuki, A; Saito, H; Yanaihara, T; Arai, Y; Oguchi, K

    1996-05-01

    To study the role of decidual monoamine oxidase (MAO)-A and -B activities before delivery, the relationship between MAO activity in fetal membranes and catecholamine (CA) concentration in amniotic fluid (AF) was determined. Fetal membranes and AF were obtained at the time of elective Cesarean section (CS group, n = 11) and Cesarean section due to fetal distress without labor pains (FD group, n = 5). MAO-A and -B activities were radiometrically measured using 14C-5-hydroxytriptamine for MAO-A substrate and 14C-benzylamine for MAO-B substrate. CA concentrations in AF were measured by high performance liquid chromatograph with an electro-chemical detector. Both MAO-A and -B activities in decidua obtained from CS were significantly lower than those obtained from FD. Both norepinephrine (NE) and epinephrine (EP) concentrations were significantly lower in the CS group than the FD group. A significant positive correlation between decidual MAO-A activity and NE concentration in AF was observed. No significant correlation was observed between MAO-B activity and the concentration of NE in AF. There was no correlation between EP concentrations and MAO activities. These results suggest that CA concentration in AF may be related to the activity of MAO in fetal membranes, determined by certain physiological processes during pregnancy. It has been suggested that metabolism of monoamines in fetal membranes also plays an important role in reducing monoamine influx into maternal myometrium from the AF.

  8. Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

    Directory of Open Access Journals (Sweden)

    Simon Burgener

    2017-12-01

    Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

  9. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

    Science.gov (United States)

    Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

    2016-11-21

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

  10. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    Science.gov (United States)

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  11. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    OpenAIRE

    Zia, Muhammad Anjum; Riaz, Ayesha; Rasul, Samreen; Abbas, Rao Zahid

    2013-01-01

    This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, ...

  12. Isolation, Identification, and Xanthine Oxidase Inhibition Activity of Alkaloid Compound from Peperomia pellucida

    Science.gov (United States)

    Fachriyah, E.; Ghifari, M. A.; Anam, K.

    2018-04-01

    The research of the isolation and xanthine oxidation inhibition activity of alkaloid compound from Peperomia pellucida has been carried out. Alkaloid extract is isolated by column chromatography and preparative TLC. Alkaloid isolate is identified spectroscopically by UV-Vis spectrophotometer, FT-IR, and LC-MS/MS. Xanthine oxidase inhibition activity is carried out by in vitro assay. The result showed that the alkaloid isolated probably has piperidine basic structure. The alkaloid isolate has N-H, C-H, C = C, C = O, C-N, C-O-C groups and the aromatic ring. The IC50 values of ethanol and alkaloid extract are 71.6658 ppm and 76.3318 ppm, respectively. Alkaloid extract of Peperomia pellucida showed higher activity than ethanol extract.

  13. Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

    Science.gov (United States)

    Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

    2013-06-01

    N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  14. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  15. Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

    Science.gov (United States)

    Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

    2013-11-01

    The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

  16. The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

    Science.gov (United States)

    Jadhao, Arun G; Biswas, Saikat P; Bhoyar, Rahul C; Pinelli, Claudia

    2017-04-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Structure–Activity Relationship of Xanthones as Inhibitors of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Ling-Yun Zhou

    2018-02-01

    Full Text Available Polygala plants contain a large number of xanthones with good physiological activities. In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study of the chemical composition of the other species Polygala sibirica led to the separation of two new xanthones—3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A and 6-O-β-d-glucopyranosyl-1,7-dimethoxy xanthone (C—together with 14 known xanthones. Among them, some xanthones have a certain xanthine oxidase (XO inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were selected to develop three-dimensional quantitative structure–activity relationship (3D-QSAR using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric, electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided insight into the structural requirements for the development of more active XO inhibitors.

  18. PPARα agonist fenofibrate protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress and MAPK activity

    International Nuclear Information System (INIS)

    Hou, Xiaoyang; Shen, Ying H.; Li, Chuanbao; Wang, Fei; Zhang, Cheng; Bu, Peili; Zhang, Yun

    2010-01-01

    Oxidative stress has been shown to play an important role in the development of hypertensive renal injury. Peroxisome proliferator-activated receptors α (PPARα) has antioxidant effect. In this study, we demonstrated that fenofibrate significantly reduced proteinuria, inflammatory cell recruitment and extracellular matrix (ECM) proteins deposition in the kidney of SHRs without apparent effect on blood pressure. To investigate the mechanisms involved, we found that fenofibrate treatment markedly reduced oxidative stress accompanied by reduced activity of renal NAD(P)H oxidase, increased activity of Cu/Zn SOD, and decreased phosphorylation of p38MAPK and JNK in the kidney of SHRs. Taken together, fenofibrate treatment can protect against hypertensive renal injury without affecting blood pressure by inhibiting inflammation and fibrosis via suppression of oxidative stress and MAPK activity.

  19. Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

    Science.gov (United States)

    Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

    2010-05-01

    Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching.

  20. Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

    Science.gov (United States)

    Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

    1997-03-25

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

  1. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    International Nuclear Information System (INIS)

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W.

    2007-01-01

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10 4 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions

  2. Natriuretic peptide receptor-C activation attenuates angiotensin II-induced enhanced oxidative stress and hyperproliferation of aortic vascular smooth muscle cells.

    Science.gov (United States)

    Madiraju, Padma; Hossain, Ekhtear; Anand-Srivastava, Madhu B

    2018-02-07

    We showed previously that natriuretic peptide receptor-C (NPR-C) agonist, C-ANP 4-23 , attenuated the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) through the inhibition of enhanced oxidative stress. Since the enhanced levels of endogenous angiotensin II (Ang II) contribute to the overexpression of Giα proteins and augmented oxidative stress in VSMC from SHR, the present study was undertaken to investigate if C-ANP 4-23 could also attenuate angiotensin II (Ang II)-induced oxidative stress and associated signaling. Ang II treatment of aortic VSMC augmented the levels of superoxide anion (O 2 - ), NADPH oxidase activity, and the expression of NADPH oxidase subunits and C-ANP 4-23 treatment attenuated all these to control levels. In addition, Ang II-induced enhanced levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl content were also attenuated toward control levels by C-ANP 4-23 treatment. On the other hand, Ang II inhibited the levels of nitric oxide (NO) and augmented the levels of peroxynitrite (OONO - ) in VSMC which were restored to control levels by C-ANP 4-23 treatment. Furthermore, C-ANP 4-23 treatment attenuated Ang II-induced enhanced expression of Giα proteins, phosphorylation of p38, JNK, and ERK 1,2 as well as hyperproliferation of VSMC as determined by DNA synthesis, and metabolic activity. These results indicate that C-ANP 4-23 , via the activation of NPR-C, attenuates Ang II-induced enhanced nitroxidative stress, overexpression of Giα proteins, increased activation of the p38/JNK/ERK 1,2 signaling pathways, and hyperproliferation of VSMC. It may be suggested that C-ANP 4-23 could be used as a therapeutic agent in the treatment of vascular remodeling associated with hypertension and atherosclerosis.

  3. The peroxidase and oxidase-like activity of NiCo{sub 2}O{sub 4} mesoporous spheres: Mechanistic understanding and colorimetric biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Su, Li, E-mail: suli@htu.edu.cn [Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Henan Key Laboratory of Green Chemical Media and Reactions, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007 (China); Dong, Wenpei; Wu, Chengke; Gong, Yijun; Zhang, Yan; Li, Ling; Mao, Guojiang; Feng, Suling [Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Henan Key Laboratory of Green Chemical Media and Reactions, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007 (China)

    2017-01-25

    The synthesized NiCo{sub 2}O{sub 4} mesoporous spheres (MS) displayed intrinsic peroxidase and oxidase-like activity were firstly reported. The catalytic mechanism of the oxidase-like activity of NiCo{sub 2}O{sub 4} MS was analyzed in detail using the electron spin resonance (ESR) method. It is found that NiCo{sub 2}O{sub 4} MS could directly oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) but did not produce {sup 1}O{sub 2} and ·OH. And the mechanism of the peroxidase-like activity of NiCo{sub 2}O{sub 4} MS was also verified that the oxidation of TMB stemmed from not only ·OH but also {sup 1}O{sub 2}. Based on the NiCo{sub 2}O{sub 4} MS showed excellent peroxidase-like activity over a broad temperature range, especially at normal body temperature, a detection tool was designed for glucose determination in diabetics' serum samples. And this detection method based on NiCo{sub 2}O{sub 4} MS gave a lower limit of detection than the method using Co{sub 3}O{sub 4} NPs and NiO NPs, as the single-component oxides of NiCo{sub 2}O{sub 4}. Our study may open up the possibility to make a great influence on the next generation of enzyme mimetics system. - Highlights: • NiCo{sub 2}O{sub 4} MS were found to possess the peroxidase and oxidase-like activity. • The peroxidase-like activity of NiCo{sub 2}O{sub 4} MS was stemmed from not only ·OH but also {sup 1}O{sub 2}. • The oxidase-like activity may stem from NiCo{sub 2}O{sub 4} MS′ oxidation rather than ·OH and {sup 1}O{sub 2}. • A colorimetric detection tool is designed for glucose determination in serum samples.

  4. Engineering glucose oxidase to minimize the influence of oxygen on sensor response

    International Nuclear Information System (INIS)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2014-01-01

    Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations

  5. NADPH- Diaphorase positive cardiac neurons in the atria of mice. A morphoquantitative study

    Directory of Open Access Journals (Sweden)

    Castelucci Patrícia

    2006-02-01

    Full Text Available Abstract Background The present study was conducted to determine the location, the morphology and distribution of NADPH-diaphorase positive neurons in the cardiac nerve plexus of the atria of mice (ASn. This plexus lies over the muscular layer of the atria, dorsal to the muscle itself, in the connective tissue of the subepicardium. NADPH- diaphorase staining was performed on whole-mount preparations of the atria mice. For descriptive purposes, all data are presented as means ± SEM. Results The majority of the NADPH-diaphorase positive neurons were observed in the ganglia of the plexus. A few single neurons were also observed. The number of NADPH-d positive neurons was 57 ± 4 (ranging from 39 to 79 neurons. The ganglion neurons were located in 3 distinct groups: (1 in the region situated cranial to the pulmonary veins, (2 caudally to the pulmonary veins, and (3 in the atrial groove. The largest group of neurons was located cranially to the pulmonary veins (66.7%. Three morphological types of NADPH-diaphorase neurons could be distinguished on the basis of their shape: unipolar cells, bipolar cells and cells with three processes (multipolar cells. The unipolar neurons predominated (78.9%, whereas the multipolar were encountered less frequently (5,3%. The sizes (area of maximal cell profile of the neurons ranged from about 90 μm2to about 220 μm2. Morphometrically, the three types of neurons were similar and there were no significant differences in their sizes. The total number of cardiac neurons (obtained by staining the neurons with NADH-diaphorase method was 530 ± 23. Therefore, the NADPH-diaphorase positive neurons of the heart represent 10% of the number of cardiac neurons stained by NADH. Conclusion The obtained data have shown that the NADPH-d positive neurons in the cardiac plexus of the atria of mice are morphologically different, and therefore, it is possible that the function of the neurons may also be different.

  6. Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

    Science.gov (United States)

    Murata, Kazuya; Nakao, Kikuyo; Hirata, Noriko; Namba, Kensuke; Nomi, Takao; Kitamura, Yoshihisa; Moriyama, Kenzo; Shintani, Takahiro; Iinuma, Munekazu; Matsuda, Hideaki

    2009-07-01

    The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.

  7. Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707

    Directory of Open Access Journals (Sweden)

    Federica Sandri

    2017-06-01

    Full Text Available Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(caa3 type oxidases (Caa3 and Ccaa3, two cbb3 type oxidases (Cbb31 and Cbb32, and one bd type cyanide-insensitive quinol oxidase (CIO. While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(caa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

  8. Molecular mechanism of voltage sensing in voltage-gated proton channels

    Science.gov (United States)

    Rebolledo, Santiago; Perez, Marta E.

    2013-01-01

    Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels. PMID:23401575

  9. Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

    Science.gov (United States)

    Im, Michelle; Dagnino, Lina

    2018-03-02

    The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.

  10. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

    Directory of Open Access Journals (Sweden)

    F.E. Sluse

    2000-03-01

    Full Text Available Cyanide-resistant alternative oxidase (AOX is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX and the proton electrochemical gradient energy-dissipating pathway (UCP lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation. Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

  12. An assay of optimal cytochrome c oxidase activity in fish gills.

    Science.gov (United States)

    Hu, Yau-Chung; Chung, Meng-Han; Lee, Tsung-Han

    2018-07-15

    Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 μg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action.

    Science.gov (United States)

    Toufexi, Eirini; Dailianis, Stefanos; Vlastos, Dimitris; Manariotis, Ioannis D

    2016-06-01

    The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10mgL(-1)) were treated under different ultrasound operating conditions (frequency at 582 and 862kHz, electric power density at 133 and 167W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/(.)O2(-), nitric oxides such as NO2(-) and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1h to 5, 10 and 100ngL(-1) and 1, 10 and 20μgL(-1) of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10μΜ L-NAME, a NO synthase inhibitor and 10μΜ DPI, a NADPH oxidase inhibitor. According to the results, 350mL of 2mgL(-1) DCF showed higher degradation (>50%) under 167W electric power density and frequency at 862kHz for 120min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of (.)O2(-) and NO2(-) generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase

  14. IAA oxidase activity in relation to adventitious root formation on stem cuttings of some forest tree species. [Salix tetrasperma, Populus Robusta, Hibiscus rosa-sinensis, Eucalyptus citriodora

    Energy Technology Data Exchange (ETDEWEB)

    Bansal, M.P.; Nanda, K.K.

    1981-01-01

    In rooting tests with stem cuttings, IAA oxidase activity was found to be very high in Salix tetrasperma and Populus 'Robusta' both of which rooted profusely, less in Hibiscus rosa-sinensis which rooted but weakly and insignificant in Eucalyptus citriodora, which did not root at all. Proteins extracted from the stem cuttings of E. citriodora inhibited IAA oxidase activity, and also root formation on hypocotyl cuttings of Phaseolus mungo.

  15. Temperature dependence of the activity of polyphenol peroxidases and polyphenol oxidases in modern and buried soils

    Science.gov (United States)

    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. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    Tarchonanthus camphoratus (camphor bush) has been widely used for numerous medicinal purposes. The aim of the present study was to evaluate the antioxidant properties, cytotoxicity and monoamine oxidase inhibition activities of the crude dichloromethane leaf extract of T. camphoratus. The antioxidant activities were ...

  17. TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

    Science.gov (United States)

    Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

    2016-01-01

    -catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls. Compared to the non-lasered control, TNF-α and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (pROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-α treatment increased VEGF expression (pROS-dependent activation of β-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-α, and ROS in RPE cells.

  18. Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

    Directory of Open Access Journals (Sweden)

    Yuno Lee

    Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

  19. Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.E.

    1979-01-01

    The distribution of catalase, amino acid oxidase, α-hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based

  20. Psoralea corylifolia L. Attenuates Nonalcoholic Steatohepatitis in Juvenile Mouse

    Directory of Open Access Journals (Sweden)

    Lishan Zhou

    2017-11-01

    Full Text Available Psoralea corylifolia L. (PC is a traditional Chinese herb used to treat yang deficiency of the spleen and kidney in pediatric disease. Recent studies have shown its liver protection and anti-oxidative effects. The aim of this study was to explore the effect and mechanism of PC on nonalcoholic steatohepatitis in juvenile mice. The juvenile mouse model of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH was established by being fed a high-fat diet in maternal-offspring manner. PC granules were prepared and the quality was assessed. The main components were identified by high performance liquid chromatography. Then, different dosages of PC were administered for 6 weeks. Homeostatic model assessment of insulin resistance, plasma liver enzymes, hepatic morphology, hepatic superoxide anion, and triglyceride/total cholesterol levels were examined. The changes of nuclear factor-κB (NF-κB activity phosphatidylinositol 3 kinase (PI3K/protein kinase B (Akt and protein kinase C-α (PKC-α/nicotinamide-adenine dinucleotide phosphate (NADPH oxidase signaling pathways in hepatic tissues were also determined. Our data demonstrated that PC significantly improved liver dysfunction, liver triglyceride/total cholesterol accumulation and insulin resistance in juvenile NAFLD/NASH mice. PC also alleviated hepatic steatosis, inflammatory cell infiltration, and fibroplasia in the portal area. Additionally, PC inhibited the activation of NF-κB and the mRNA expression of inflammatory factors while enhancing PI3K/Akt signaling in hepatic tissues. PC could also reduce hepatic superoxide anion levels, and NADPH oxidase activity as well as p47phox protein expression and PKCα activation in hepatic tissues. The results suggest that PC is effective in the treatment of NASH in juvenile mice. The mechanism may be related to the attenuation of hepatic oxidative stress through the PKC-α/NADPH oxidase signaling pathway.

  1. Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions

    NARCIS (Netherlands)

    Glogauer, Michael; Marchal, Christophe C.; Zhu, Fei; Worku, Aelaf; Clausen, Björn E.; Foerster, Irmgard; Marks, Peter; Downey, Gregory P.; Dinauer, Mary; Kwiatkowski, David J.

    2003-01-01

    Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To

  2. Crystal Structure of Perakine Reductase, Founding Member of a Novel Aldo-Keto Reductase (AKR) Subfamily That Undergoes Unique Conformational Changes during NADPH Binding*

    Science.gov (United States)

    Sun, Lianli; Chen, Yixin; Rajendran, Chitra; Mueller, Uwe; Panjikar, Santosh; Wang, Meitian; Mindnich, Rebekka; Rosenthal, Cindy; Penning, Trevor M.; Stöckigt, Joachim

    2012-01-01

    Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His6-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His6-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α8/β6 barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family. PMID:22334702

  3. Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

    Science.gov (United States)

    Gabler, M; Fischer, L

    1999-08-01

    The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

  4. Lead-elevated activity of xanthine oxidase in lead-exposed workers

    Directory of Open Access Journals (Sweden)

    Sławomir Kasperczyk

    2013-04-01

    Full Text Available Background: The aim of the present study was to explore the connection between lead toxicity and the activity of xanthine oxidase (XO. In addition, we indicated the uric acid (UA and creatinine levels and concentration of erythrocyte malondialdehyde (MDA to estimate oxidative stress intensity. Materials and Methods: The examined group consisted of 125 healthy male employees of zinc and lead works. The examined group was divided into tertiles according to blood lead levels. In the collected blood samples, concentrations of lead-exposure indices, UA, creatinine, and MDA as well as activity of XO were measured concomitantly. The control group consisted of 32 healthy male administrative workers who were exposed to lead only environmentally. Results: XO activity and MDA level were significantly elevated in all tertiles compared to the control group. Creatinine level was significantly elevated in the medium and high tertiles. However, the level of UA was significantly elevated in the high tertile, while in the low and medium tertile only a tendency toward higher values was observed. Conclusions: Occupational exposure to lead induces activity of XO. This induction may contribute to the observed simultaneously increased oxidative stress, measured as MDA level, and the increased level of UA. Med Pr 2013;64(2:175–180

  5. A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions

    NARCIS (Netherlands)

    Patel, H. R.; Frederiks, W. M.; Marx, F.; Best, A. J.; van Noorden, C. J.

    1991-01-01

    The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between

  6. cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

    Czech Academy of Sciences Publication Activity Database

    Černý, Ondřej; Anderson, K.E.; Stephens, L.R.; Hawkins, P.T.; Šebo, Peter

    2017-01-01

    Roč. 198, č. 3 (2017), s. 1285-1296 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GA13-14547S; GA MŠk(CZ) LM2015064 Institutional support: RVO:61388971 Keywords : BORDETELLA-PERTUSSIS * NADPH OXIDASE * CYCLIC-AMP Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.856, year: 2016

  7. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein......+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n......M) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production...

  8. SHIP-1 Increases Early Oxidative Burst and Regulates Phagosome Maturation in Macrophages1

    Science.gov (United States)

    Kamen, Lynn A.; Levinsohn, Jonathan; Cadwallader, Amy; Tridandapani, Susheela; Swanson, Joel A.

    2010-01-01

    Although the inositol phosphatase SHIP-1 is generally thought to inhibit signaling for Fc receptor-mediated phagocytosis, the product of its activity, phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2) has been implicated in activation of the NADPH oxidase. This suggests that SHIP-1 positively regulates generation of reactive oxygen species after phagocytosis. To examine how SHIP-1 activity contributes to Fc receptor-mediated phagocytosis, we measured and compared phospholipid dynamics, membrane trafficking and the oxidative burst in macrophages from SHIP-1-deficient and wild-type mice. SHIP-1-deficient macrophages showed significantly elevated ratios of PI(3,4,5) P3 to PI(3,4)P2 on phagosomal membranes. Imaging reactive oxygen intermediate activities in phagosomes revealed decreased early NADPH oxidase activity in SHIP-1-deficient macrophages. SHIP-1-deficiency also altered later stages of phagosome maturation, as indicated by the persistent elevation of PI(3)P and the early localization of Rab5a to phagosomes. These direct measurements of individual organelles indicate that phagosomal SHIP-1 enhances the early oxidative burst through localized alteration of the membrane 3′ phosphoinositide composition. PMID:18490750

  9. β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade.

    Science.gov (United States)

    Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A; Hamilton, Elisha J; Figtree, Gemma A; Rasmussen, Helge H

    2015-09-01

    Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes. Copyright © 2015 the American Physiological Society.

  10. D-amino acid oxidase activator gene (DAOA) variation affects cerebrospinal fluid homovanillic acid concentrations in healthy Caucasians

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas

    2012-01-01

    The D-amino acid oxidase activator (DAOA) protein regulates the function of D-amino oxidase (DAO), an enzyme that catalyzes the oxidative deamination of D-3,4-dihydroxyphenylalanine (D-DOPA) and D-serine. D-DOPA is converted to L-3,4-DOPA, a precursor of dopamine, whereas D-serine participates...... in glutamatergic transmission. We hypothesized that DAOA polymorphisms are associated with dopamine, serotonin and noradrenaline turnover in the human brain. Four single-nucleotide polymorphisms, previously reported to be associated with schizophrenia, were genotyped. Cerebrospinal fluid (CSF) samples were drawn...... by lumbar puncture, and the concentrations of the major dopamine metabolite homovanillic acid (HVA), the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the major noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. Two of the investigated polymorphisms, rs...

  11. Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

    Directory of Open Access Journals (Sweden)

    Ashutosh Kumar

    2018-05-01

    Full Text Available Exposure to (bisulfite (HSO3– and sulfite (SO32– has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bisulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–, peroxymonosulfate (–O3SOO., and especially the sulfate (SO4. – anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO, which has been shown to form protein radicals. Although formation of (bisulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bisulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bisulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bisulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bisulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bisulfite-derived protein radical formation and show the involvement of

  12. Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

    International Nuclear Information System (INIS)

    Liu Jingquan; Paddon-Row, Michael N.; Gooding, J. Justin

    2006-01-01

    An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase - a probable consequence of the FAD being immersed in a protein environment - was responsible for the inability of the enzyme to be turned over under anaerobic conditions

  13. Increased xanthine oxidase during labour--implications for oxidative stress.

    Science.gov (United States)

    Many, A; Roberts, J M

    1997-11-01

    Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women (P = 0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

  14. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Lages, Nuno; Oldiges, M.

    2009-01-01

    to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...... oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol...

  15. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

    DEFF Research Database (Denmark)

    Nissen, T.L.; Anderlund, M.; Nielsen, Jens

    2001-01-01

    The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/ADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can...... cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein...

  16. Protection against Ischemia-Induced Oxidative Stress Conferred by Vagal Stimulation in the Rat Heart: Involvement of the AMPK-PKC Pathway

    Directory of Open Access Journals (Sweden)

    Wei-Jin Zang

    2012-11-01

    Full Text Available Reactive oxygen species (ROS production is an important mechanism in myocardial ischemia and nicotinamide adenine dinucleotide phosphate (NADPH oxidase is one of major sources of ROS in the heart. Previous studies showed that vagus nerve stimulation (VNS is beneficial in treating ischemic heart diseases. However, the effect of VNS on ROS production remains elusive. In this study, we investigated the role of VNS onischemia-induced ROS production. Our results demonstrated that VNS alleviated the myocardial injury, attenuated the cardiac dysfunction, reserved the antioxidant enzyme activity and inhibited the formation of ROS as evidenced by the decreased NADPH oxidase (Nox activity and superoxide fluorescence intensity as well as the expression of p67phox, Rac1 and nitrotyrosine. Furthermore, VNS resulted in the phosphorylation and activation of adenosine monophosphate activated protein kinase (AMPK, which in turn led to an inactivation of Nox by protein kinase C (PKC; however, the phenomena were repressed by the administration of a muscarinic antagonist atropine. Taken together, these data indicate that VNS decreases ROS via AMPK-PKC-Nox pathway; this may have potential importance for the treatment of ischemic heart diseases.

  17. The activity of ascorbic acid and catechol oxidase, the rate of photosynthesis and respiration as related to plant organs, stage of development and copper supply

    Directory of Open Access Journals (Sweden)

    St. Łyszcz

    2015-06-01

    Full Text Available Some experiments were performed to investigate the physiological role of copper in oat and sunflower and to recognize some effects of copper deficiency. Oat and sunflower plants were grown in pots on a peat soil under copper deficiency conditions (–Cu or with the optimal copper supply (+Cu. In plants the following measurements were carried out: 1 the activity of ascorbic acid oxidase (AAO and of catechol oxidase (PPO in different plant organs and at different stages of plant development, 2 the activity and the rate of photosynthesis, 3 the activity of RuDP-carboxylase, 4 the intensity of plant respiration. The activity of AAO and of PPO, and also the rate and the activity of photosynthesis were significantly lower under conditions of copper deficiency. The activity of both discussed oxidases depended on: 1 the plant species, 2 plant organs, 3 stage of plant development. Copper deficiency caused decrease of the respiration intensity of sunflower leaves but it increased to some extent the respiration of oat tops. Obtained results are consistent with the earlier suggestion of the authors that the PPO activity in sunflower leaves could be a sensitive indicator of copper supply of the plants, farther experiments are in progress.

  18. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

    Science.gov (United States)

    Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

    2018-01-01

    Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  19. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Zheng Lu

    2018-05-01

    Full Text Available Arctigenin (ARG, one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21 and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α and transforming growth factor beta1 (TGF-β1 in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126. Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  20. Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway.

    Science.gov (United States)

    Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

    2018-01-01

    Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L. , has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

  1. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  2. Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2013-11-01

    Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

  3. Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

    Science.gov (United States)

    Thrower, J S; Blalock, R; Klinman, J P

    2001-08-14

    1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

  4. Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

    Directory of Open Access Journals (Sweden)

    Larissa Dyugovskaya

    2016-01-01

    Full Text Available Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b+/CD63+/MPO+/LC3B+ and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH (29 cycles/day for 5 days completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI, a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC abrogated the effects of IH by inducing large CD66b+/LC3B+ Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

  5. Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kolaríková, Katerina; Galuszka, Petr; Sedlárová, Iva; Sebela, Marek; Frébort, Ivo

    2009-01-01

    The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

  6. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (Fc epsilon RI) Signaling in Mast Cells by Suppressing the Function of Fc epsilon RI-Cholesterol Signalosome

    Czech Academy of Sciences Publication Activity Database

    Dráberová, Lubica; Paulenda, Tomáš; Hálová, Ivana; Potůčková, Lucie; Bugajev, Viktor; Bambousková, Monika; Tůmová, Magda; Dráber, Petr

    2015-01-01

    Roč. 10, č. 12 (2015), e0144596-e0144596 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MŠk LD12073 Institutional support: RVO:68378050 Keywords : KAPPA-B Activation * Lipid rafts * Nadph oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.057, year: 2015

  7. Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Alessandro R. Ferrari

    2018-01-01

    Full Text Available The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615 and XP_003665713 (MtVAO713, were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

  8. [Signaling Systems of Rhizobia (Rhizobiaceae) and Leguminous Plants (Fabaceae) upon the Formation of a Legume-Rhizobium Symbiosis (Review)].

    Science.gov (United States)

    Glyan'ko, A K

    2015-01-01

    Data from the literature and our own data on the participation and interrelation of bacterial signaling Nod-factors and components of the calcium, NADPH-oxidase, and NO-synthase signaling systems of a plant at the preinfection and infectious stages of the formation of a legume-rhizobium symbiosis are summarized in this review. The physiological role of Nod-factors, reactive oxygen species (ROS), calcium (Ca2+), NADPH-oxidase, nitric oxide (NO), and their cross influence on the processes determining the formation of symbiotic structures on the roots of the host plant is discussed.

  9. Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42

    OpenAIRE

    El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M.; El-Ewasy, Sara M.

    2016-01-01

    Background Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. Results One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis...

  10. In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

    Science.gov (United States)

    Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

    2010-11-15

    In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

  11. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    OpenAIRE

    Jänne, J; Morris, D R

    1984-01-01

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

  12. Cytochrome oxidase as an indicator of ice storage and frozen storage

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2001-01-01

    in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

  13. Antiglycation, radical scavenging, and semicarbazide-sensitive amine oxidase inhibitory activities of acetohydroxamic acid in vitro

    Directory of Open Access Journals (Sweden)

    Liu YH

    2017-07-01

    Full Text Available Yuh-Hwa Liu,1,2,* Yeh-Lin Lu,3,* Der-Zen Liu,4 Wen-Chi Hou5 1Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; 2Department of General Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3Department of Pharmacy, Taipei Medical University, Taipei, Taiwan; 4Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan; 5Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan *These authors contributed equally to this work Abstract: Advanced glycation endproducts (AGEs can promote intracellular reactive oxygen species production, and the levels of AGEs are highly correlated with cardiovascular disease and diabetes complications. Acetohydroxamic acid (acetH is a bacterial urease inhibitor drug used to treat kidney stones and infections in the urinary tract, and hydroxyurea (HU is a drug used for antineoplasm and sickle cell diseases. Both acetH and HU are hydroxamic acid derivatives. It was found that acetH and HU at 2.5 or 5 mM showed anti-AGE formation by lowering the AGEs’ fluorescent intensities and Nε-(carboxymethyllysine formation in bovine serum albumin/galactose models, and both showed better and significant differences (P<0.05 compared to the positive control of aminoguanidine. Regarding radical scavenging activities, the half-inhibition concentrations (IC50 of acetH against α,α-diphenyl-β-picrylhydrazyl radical and hydroxyl radical were 34.86 and 104.42 µM, respectively. The IC50 of acetH against semicarbazide-sensitive amine oxidase was 10.56 µM, and acetH showed noncompetitive inhibition respective to the substrates (benzylamine. The antiglycation, antioxidant, and semicarbazide-sensitive amine oxidase inhibitory activities of acetH prove that it has the potential for treating cardiovascular disease and diabetes complications and it needs further investigation in animal models. Keywords: acetH, AGEs, Nε

  14. Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

    Science.gov (United States)

    Nguyen, Don Trinh; Göpfert, Jens Christian; Ikezawa, Nobuhiro; MacNevin, Gillian; Kathiresan, Meena; Conrad, Jürgen; Spring, Otmar; Ro, Dae-Kyun

    2010-01-01

    Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. PMID:20351109

  15. Increase in intracellular free/bound NAD[P]H as a cause of Cd-induced oxidative stress in the HepG2 cells

    International Nuclear Information System (INIS)

    Yang, M.S.; Li, D.; Lin, T.; Zheng, J.J.; Zheng, W.; Qu, J.Y.

    2008-01-01

    The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG 2 cells at 0.5, 1.5, 3 and 4.5 h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5 h after treatment while the change in FAD/NAD[P]H did not occur until 3 h after exposure. Production of ROS was also increased at 1.5 h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5 h and remained elevated until 4.5 h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events

  16. [Effects of melaxen and valdoxan on the activity of glutathione antioxidant system and NADPH-producing enzymes in rat heart under experimental hyperthyroidism conditions].

    Science.gov (United States)

    Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

    2013-01-01

    The effects of melaxen and valdoxan on the activity of glutathione antioxidant system and some NADPH-producing enzymes have been studied under conditions of experimental hyperthyroidism in rat heart. Under the action of these drugs, reduced glutathione (GSH) content increased as compared to values observed under the conditions of pathology. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP), glucose-6-phosphate dehydrogenase, and NADP isocitrate dehydrogenase (increased under pathological conditions) change toward the intact control values upon the introduction of both drugs. The influence of melaxen and valdoxan, capable of producing antioxidant effect, leads apparently to the inhibition of free-radical oxidation processes and, as a consequence, the reduction of mobilization degree of the glutathione antioxidant system.

  17. Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

    Directory of Open Access Journals (Sweden)

    Sluse F.E.

    1998-01-01

    Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

  18. Activation of α-7 nicotinic acetylcholine receptor reduces ischemic stroke injury through reduction of pro-inflammatory macrophages and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Zhenying Han

    Full Text Available Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1 and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist, methyllycaconitine (MLA, nAchR antagonist, or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO. Behavior test, lesion volume, CD68(+, M1 (CD11b(+/Iba1(+ and M2 (CD206/Iba1+ microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68(+ and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect.

  19. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

    Directory of Open Access Journals (Sweden)

    Tom A. Ewing

    2018-01-01

    Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

  20. A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

    Science.gov (United States)

    Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

    2018-01-14

    Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.