NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

PubMed

Seo, Ji-Seon; Park, Jin-Young; Choi, Juli; Kim, Tae-Kyung; Shin, Joo-Hyun; Lee, Ja-Kyeong; Han, Pyung-Lim

2012-07-11

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

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

PubMed

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

2014-10-01

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM 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.

α-Synuclein stimulation of monoamine oxidase-B and legumain protease mediates the pathology of Parkinson's disease.

PubMed

Kang, Seong Su; Ahn, Eun Hee; Zhang, Zhentao; Liu, Xia; Manfredsson, Fredric P; Sandoval, Ivette M; Dhakal, Susov; Iuvone, P Michael; Cao, Xuebing; Ye, Keqiang

2018-06-15

Dopaminergic neurodegeneration in Parkinson's disease (PD) is associated with abnormal dopamine metabolism by MAO-B (monoamine oxidase-B) and intracellular α-Synuclein (α-Syn) aggregates, called the Lewy body. However, the molecular relationship between α-Syn and MAO-B remains unclear. Here, we show that α-Syn directly binds to MAO-B and stimulates its enzymatic activity, which triggers AEP (asparagine endopeptidase; legumain) activation and subsequent α-Syn cleavage at N103, leading to dopaminergic neurodegeneration. Interestingly, the dopamine metabolite, DOPAL, strongly activates AEP, and the N103 fragment of α-Syn binds and activates MAO-B. Accordingly, overexpression of AEP in SNCA transgenic mice elicits α-Syn N103 cleavage and accelerates PD pathogenesis, and inhibition of MAO-B by Rasagiline diminishes α-Syn-mediated PD pathology and motor dysfunction. Moreover, virally mediated expression of α-Syn N103 induces PD pathogenesis in wild-type, but not MAO-B-null mice. Our findings thus support that AEP-mediated cleavage of α-Syn at N103 is required for the association and activation of MAO-B, mediating PD pathogenesis. © 2018 The Authors.

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells*

PubMed Central

Shah, Anu; Xia, Ling; Goldberg, Howard; Lee, Ken W.; Quaggin, Susan E.; Fantus, I. George

2013-01-01

Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN. PMID:23329835

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

PubMed

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.

Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis

PubMed Central

Wu, Jason Boyang; Shao, Chen; Li, Xiangyan; Li, Qinlong; Hu, Peizhen; Shi, Changhong; Li, Yang; Chen, Yi-Ting; Yin, Fei; Liao, Chun-Peng; Stiles, Bangyan L.; Zhau, Haiyen E.; Shih, Jean C.; Chung, Leland W.K.

2014-01-01

Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa. PMID:24865426

Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis.

PubMed

Wu, Jason Boyang; Shao, Chen; Li, Xiangyan; Li, Qinlong; Hu, Peizhen; Shi, Changhong; Li, Yang; Chen, Yi-Ting; Yin, Fei; Liao, Chun-Peng; Stiles, Bangyan L; Zhau, Haiyen E; Shih, Jean C; Chung, Leland W K

2014-07-01

Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa.

Cysteine-mediated aggregation of Au nanoparticles: the development of a H2O2 sensor and oxidase-based biosensors.

PubMed

Wang, Fuan; Liu, Xiaoqing; Lu, Chun-Hua; Willner, Itamar

2013-08-27

The cysteine-stimulated aggregation of Au nanoparticles (Au NPs) is used as an auxiliary reporting system for the optical detection of H2O2, for optical probing of the glucose oxidase (GOx) and the catalyzed oxidation of glucose, for probing the biocatalytic cascade composed of acetylcholine esterase/choline oxidase (AChE/ChOx), and for following the inhibition of AChE. The analytical paradigm is based on the I(-)-catalyzed oxidation of cysteine by H2O2 to cystine, a process that prohibits the cysteine-triggered aggregation of the Au NPs. The system enabled the analysis of H2O2 with a detection limit of 2 μM. As the GOx-biocatalyzed oxidation of glucose yields H2O2, and the AChE/ChOx cascade leads to the formation of H2O2, the two biocatalytic processes could be probed by the cysteine-stimulated aggregation of the Au NPs. Since AChE is inhibited by 1,5-bis(4-allyldimethylammonium phenyl)pentane-3-one dibromide, the biocatalytic AChE/ChOx cascade is inhibited by the inhibitor, thus leading to the enhanced cysteine-mediated aggregation of the NPs. The results suggest the potential implementation of the cysteine-mediated aggregation of Au NPs in the presence of AChE/ChOx as a sensing platform for the optical detection of chemical warfare agents.

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker for Agrobacterium tumefaciens-Mediated Transformation of Maize

PubMed Central

Li, Xianggan; Volrath, Sandy L.; Nicholl, David B.G.; Chilcott, Charles E.; Johnson, Marie A.; Ward, Eric R.; Law, Marcus D.

2003-01-01

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil. PMID:12972658

NMDARs Mediate the Role of Monoamine Oxidase A in Pathological Aggression

PubMed Central

Bortolato, Marco; Godar, Sean C.; Melis, Miriam; Soggiu, Alessio; Roncada, Paola; Casu, Angelo; Flore, Giovanna; Chen, Kevin; Frau, Roberto; Urbani, Andrea; Castelli, M. Paola; Devoto, Paola; Shih, Jean C.

2012-01-01

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25–6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality. PMID:22723698

NMDARs mediate the role of monoamine oxidase A in pathological aggression.

PubMed

Bortolato, Marco; Godar, Sean C; Melis, Miriam; Soggiu, Alessio; Roncada, Paola; Casu, Angelo; Flore, Giovanna; Chen, Kevin; Frau, Roberto; Urbani, Andrea; Castelli, M Paola; Devoto, Paola; Shih, Jean C

2012-06-20

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25-6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.

NADPH oxidases in the arbuscular mycorrhizal symbiosis.

PubMed

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

NADPH oxidases in the arbuscular mycorrhizal symbiosis

PubMed Central

Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

2016-01-01

ABSTRACT Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases.

PubMed

Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong

2012-06-01

Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases. Published by Elsevier Ltd.

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress.

PubMed

Sautin, Yuri Y; Nakagawa, Takahiko; Zharikov, Sergey; Johnson, Richard J

2007-08-01

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.

Monocyte and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice

PubMed Central

Grimm, Melissa J.; Vethanayagam, R. Robert; Almyroudis, Nikolaos G.; Dennis, Carly G.; Khan, A. Nazmul H.; D’Auria, Anthony; Singel, Kelly L.; Davidson, Bruce A.; Knight, Paul R.; Blackwell, Timothy S.; Hohl, Tobias M.; Mansour, Michael K.; Vyas, Jatin M.; Röhm, Marc; Urban, Constantin F.; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H.

2013-01-01

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was more than 100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and pro-inflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wildtype mice was mild and transient. Together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species.

PubMed

Tsirmoula, Sotiria; Lamprou, Margarita; Hatziapostolou, Maria; Kieffer, Nelly; Papadimitriou, Evangelia

2015-03-01

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin alpha v beta 3 (ανβ3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPβ/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN-ανβ3 interaction abolished PTN-induced ROS generation, suggesting that ανβ3 is also involved. The latter was confirmed in CHO cells that do not express β3 or over-express wild-type β3 or mutant β3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type β3 but not in cells not expressing or expressing mutant β3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανβ3 and RPTPβ/ζ and activation of c-src, PI3K and ERK1/2 kinases. Copyright © 2015 Elsevier Inc. All rights reserved.

Electrochemical studies of biocatalytic anode of sulfonated graphene/ferritin/glucose oxidase layer-by-layer biocomposite films for mediated electron transfer.

PubMed

Inamuddin; Haque, Sufia Ul; Naushad, Mu

2016-06-01

In this study, a bioanode was developed by using layer-by-layer (LBL) assembly of sulfonated graphene (SG)/ferritin (Frt)/glucose oxidase (GOx). The SG/Frt biocomposite was used as an electron transfer elevator and mediator, respectively. Glucose oxidase (GOx) from Aspergillus niger was applied as a glucose oxidation biocatalyst. The electrocatalytic oxidation of glucose using GOx modified electrode increases with an increase in the concentration of glucose in the range of 10-50mM. The electrochemical measurements of the electrode was carried out by using cyclic voltammetry (CV) at different scan rates (20-100mVs(-1)) in 30mM of glucose solution prepared in 0.3M potassium ferrocyanide (K4Fe(CN)6) and linear sweep voltammetry (LSV). A saturation current density of 50±2mAcm(-2) at a scan rate of 100mVs(-1) for the oxidation of 30Mm glucose is achieved. Copyright © 2016 Elsevier Inc. All rights reserved.

NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

PubMed Central

Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

2017-01-01

Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

PubMed

Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

2017-07-25

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

Protection against acetaminophen-induced liver injury by allopurinol is dependent on aldehyde oxidase-mediated liver preconditioning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, C. David; McGill, Mitchell R.; Lebofsky, Margitta

2014-02-01

Acetaminophen (APAP) overdose causes severe and occasionally fatal liver injury. Numerous drugs that attenuate APAP toxicity have been described. However these compounds frequently protect by cytochrome P450 inhibition, thereby preventing the initiating step of toxicity. We have previously shown that pretreatment with allopurinol can effectively protect against APAP toxicity, but the mechanism remains unclear. In the current study, C3HeB/FeJ mice were administered allopurinol 18 h or 1 h prior to an APAP overdose. Administration of allopurinol 18 h prior to APAP overdose resulted in an 88% reduction in liver injury (serum ALT) 6 h after APAP; however, 1 h pretreatmentmore » offered no protection. APAP-cysteine adducts and glutathione depletion kinetics were similar with or without allopurinol pretreatment. The phosphorylation and mitochondrial translocation of c-jun-N-terminal-kinase (JNK) have been implicated in the progression of APAP toxicity. In our study we showed equivalent early JNK activation (2 h) however late JNK activation (6 h) was attenuated in allopurinol treated mice, which suggests that later JNK activation is more critical for the toxicity. Additional mice were administered oxypurinol (primary metabolite of allopurinol) 18 h or 1 h pre-APAP, but neither treatment protected. This finding implicated an aldehyde oxidase (AO)-mediated metabolism of allopurinol, so mice were treated with hydralazine to inhibit AO prior to allopurinol/APAP administration, which eliminated the protective effects of allopurinol. We evaluated potential targets of AO-mediated preconditioning and found increased hepatic metallothionein 18 h post-allopurinol. These data show metabolism of allopurinol occurring independent of P450 isoenzymes preconditions the liver and renders the animal less susceptible to an APAP overdose. - Highlights: • 18 h allopurinol pretreatment protects against acetaminophen-induced liver injury. • 1 h allopurinol pretreatment does not protect

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK

PubMed Central

Nomura, Johji; Busso, Nathalie; Ives, Annette; Tsujimoto, Syunsuke; Tamura, Mizuho; So, Alexander; Yamanaka, Yoshihiro

2013-01-01

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes. PMID:24086554

Reusable and Mediator-Free Cholesterol Biosensor Based on Cholesterol Oxidase Immobilized onto TGA-SAM Modified Smart Bio-Chips

PubMed Central

Rahman, Mohammed M.

2014-01-01

A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields. PMID:24949733

Activation of monoamine oxidase isotypes by prolonged intake of aluminum in rat brain.

PubMed

Huh, Jae-Wan; Choi, Myung-Min; Lee, Jang Han; Yang, Seung-Ju; Kim, Mi Jung; Choi, Jene; Lee, Kwan Ho; Lee, Jong Eun; Cho, Sung-Woo

2005-10-01

Rats were fed 100 microM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency (kcat/KM) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.

In vivo low-level light therapy increases cytochrome oxidase in skeletal muscle.

PubMed

Hayworth, Christopher R; Rojas, Julio C; Padilla, Eimeira; Holmes, Genevieve M; Sheridan, Eva C; Gonzalez-Lima, F

2010-01-01

Low-level light therapy (LLLT) increases survival of cultured cells, improves behavioral recovery from neurodegeneration and speeds wound healing. These beneficial effects are thought to be mediated by upregulation of mitochondrial proteins, especially the respiratory enzyme cytochrome oxidase. However, the effects of in vivo LLLT on cytochrome oxidase in intact skeletal muscle have not been previously investigated. We used a sensitive method for enzyme histochemistry of cytochrome oxidase to examine the rat temporalis muscle 24 h after in vivo LLLT. The findings showed for the first time that in vivo LLLT induced a dose- and fiber type-dependent increase in cytochrome oxidase in muscle fibers. LLLT was particularly effective at enhancing the aerobic capacity of intermediate and red fibers. The findings suggest that LLLT may enhance the oxidative energy metabolic capacity of different types of muscle fibers, and that LLLT may be used to enhance the aerobic potential of skeletal muscle.

Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata

PubMed Central

Ogasawara, Kimi; Yamada, Kenji; Hatsugai, Noriyuki; Imada, Chiaki; Nishimura, Mikio

2016-01-01

Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment. PMID:26867214

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

NASA Astrophysics Data System (ADS)

DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

2003-04-01

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Homocysteine facilitates LOX-1 activation and endothelial death through the PKCβ and SIRT1/HSF1 mechanism: relevance to human hyperhomocysteinaemia.

PubMed

Hung, Ching-Hsia; Chan, Shih-Hung; Chu, Pei-Ming; Tsai, Kun-Ling

2015-09-01

HHcy (hyperhomocysteinaemia) is one of the major risk factors for cardiovascular diseases. A high concentration of Hcy (homocysteine) induces endothelial dysfunction by activating endothelial oxidative stress. LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) plays a vital role in regulating the progression of atherosclerotic lesions. LOX-1 activation causes endothelial apoptosis and inflammation. The mechanism is still unclear as to whether Hcy affects human endothelial LOX-1 expression. LOX-1 expression level was confirmed by Western blotting assay in Hcy-treated endothelial cells. L-Methionine was used for HHcy induction in animals. Our results suggested that Hcy increased PKCβ (protein kinase Cβ) activation to enhance the LOX-1 expression level. The up-regulation of PKCβ phosphorylation subsequently causes ROS (reactive oxygen species) formation and SIRT1 (sirtuin 1) degradation through a proteasome-dependent mechanism, thereby mitigating the activity of SIRT1 by deacetylating HSF1 (heat-shock transcription factor 1). We also found that NOX2 is a key NAPDH oxidase isoform responsible for the Hcy-caused ROS formation. The overexpression of SIRT1 and HSF1 reduced the Hcy-induced LOX-1 activation. Silencing PKCβ function also reduced LOX-1 activation and endothelial apoptosis caused by Hcy. Our hypothesis was supported by analysing the data from methionine-induced HHcy-affected animals. Our data indicate a new direction for LOX-1 regulation by the modulation of the PKCβ/NAPDH oxidase/SIRT1/HSF1 mechanism. Our findings might provide a novel route for developing new therapeutic treatments for HHcy. © 2015 Authors; published by Portland Press Limited.

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

PubMed

Kim, Da Jung; Kim, Yong Sik

2015-01-01

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

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

PubMed Central

Kim, Da Jung; Kim, Yong Sik

2015-01-01

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

Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate.

PubMed

Liu, Cheng-Hao; Tseng, Wei-Lung

2011-10-03

This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe(3)O(4) nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H(2)O(2), the reaction pH between Fe(3)O(4) NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe(3)O(4) NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe(3)O(4) NPs and oxidase for the quantification of specific substrates through the H(2)O(2)-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase-Fe(3)O(4), galactose oxidase-Fe(3)O(4), and choline oxidase-Fe(3)O(4) composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. Copyright © 2011 Elsevier B.V. All rights reserved.

Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

PubMed Central

Bongers, Kale S.; Fox, Daniel K.; Kunkel, Steven D.; Stebounova, Larissa V.; Murry, Daryl J.; Pufall, Miles A.; Ebert, Scott M.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.

2014-01-01

Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy. PMID:25406264

Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis.

PubMed

Cathcart, Martha K

2004-01-01

Monocyte extravasation into the vessel wall has been shown to be a critical step in the development of atherosclerosis. Upon activation, monocytes produce a burst of superoxide anion due to activation of the NADPH oxidase enzyme complex. Monocyte-derived superoxide anion contributes to oxidant stress in inflammatory sites, is required for monocyte-mediated LDL oxidation, and alters basic cell functions such as adhesion and proliferation. We hypothesize that monocyte-derived superoxide anion production contributes to atherosclerotic lesion formation. In this brief review, we summarize our current understanding of the signal transduction pathways regulating NADPH oxidase activation and related superoxide anion production in activated human monocytes. Novel pathways are identified that may serve as future targets for therapeutic intervention in this pathogenic process. The contributions of superoxide anion and NADPH oxidase to atherogenesis are discussed. Future experiments are needed to clarify the exact role of NADPH oxidase-derived superoxide anion in atherogenesis, particularly that derived from monocytes.

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

PubMed Central

Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

2013-01-01

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

PubMed Central

Smesrud, Logan; Tebo, Bradley M.

2016-01-01

ABSTRACT The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the

Multi-Copper Oxidases and Human Iron Metabolism

PubMed Central

Vashchenko, Ganna; MacGillivray, Ross T. A.

2013-01-01

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

PubMed

Suroviec, Alice H

2017-01-01

The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

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

PubMed

Vokurková, M; Rauchová, H; Řezáčová, L; Vaněčková, I; Zicha, J

2015-01-01

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

Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

PubMed Central

Agwu, D E; McPhail, L C; Sozzani, S; Bass, D A; McCall, C E

1991-01-01

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway. Images

NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology

PubMed Central

Ushio-Fukai, Masuko; Malik, Asrar B.

2009-01-01

Abstract Reactive oxygen species (ROS) including superoxide (O2·−) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810. PMID:18783313

Lessons About the Pathogenesis and Management of Aspergillosis from Studies in Chronic Granulomatous Disease

PubMed Central

Gallin, John I.; Zarember, Kol

2007-01-01

Chronic Granulomatous Disease (CGD) is a rare disorder caused by mutations in the NADPH oxidase. The CGD phenotype includes granuloma formation and susceptibility to infection with microorganisms including Aspergillus. The immune adjuvant interferon-γ and the antifungal agent itraconazole have reduced the incidence of infections in CGD. Studies using CGD phagocytes have shown that reactive oxygen species (ROS), products of the NAPDH oxidase, are critical for killing Aspergillus hyphae. But despite lack of ROS production, CGD patients generally only get infected with Aspergillus after heavy exposure. To study why CGD patients are not infected with Aspergillus more frequently we studied host defense against this ubiquitous mold further. We found that neutrophil lactoferrin is fungistatic for Aspergillus fumigatus spores by chelation of iron, an essential growth factor. Thus, the neutrophil employs both nonoxidative (lactoferrin) and oxidative (hydrogen peroxide) defense mechanisms against A. fumigatus spores and hyphae, respectively. PMID:18528501

Monoamine oxidases are mediators of endothelial dysfunction in the mouse aorta.

PubMed

Sturza, Adrian; Leisegang, Matthias S; Babelova, Andrea; Schröder, Katrin; Benkhoff, Sebastian; Loot, Annemarieke E; Fleming, Ingrid; Schulz, Rainer; Muntean, Danina M; Brandes, Ralf P

2013-07-01

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease

Ethanol-induced erectile dysfunction and increased expression of pro-inflammatory proteins in the rat cavernosal smooth muscle are mediated by NADPH oxidase-derived reactive oxygen species.

PubMed

Leite, Letícia N; do Vale, Gabriel T; Simplicio, Janaina A; De Martinis, Bruno S; Carneiro, Fernando S; Tirapelli, Carlos R

2017-06-05

Ethanol consumption is associated with an increased risk of erectile dysfunction (ED), but the molecular mechanisms through which ethanol causes ED remain elusive. Reactive oxygen species are described as mediators of ethanol-induced cell toxicity/damage in distinctive tissues. The enzyme NADPH oxidase is the main source of reactive oxygen species in the endothelium and vascular smooth muscle cells and ethanol is described to increase NADPH oxidase activation and reactive oxygen species generation. This study evaluated the contribution of NADPH oxidase-derived reactive oxygen species to ethanol-induced ED, endothelial dysfunction and production of pro-inflammatory and redox-sensitive proteins in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) or ethanol plus apocynin (30mg/kg/day; p.o. gavage) for six weeks. Apocynin prevented both the decreased in acetylcholine-induced relaxation and intracavernosal pressure induced by ethanol. Ethanol increased superoxide anion (O 2 - ) generation and catalase activity in CSM, and treatment with apocynin prevented these responses. Similarly, apocynin prevented the ethanol-induced decreased of nitrate/nitrite (NOx), hydrogen peroxide (H 2 O 2 ) and SOD activity. Treatment with ethanol increased p47phox translocation to the membrane as well as the expression of Nox2, COX-1, catalase, iNOS, ICAM-1 and p65. Apocynin prevented the effects of ethanol on protein expression and p47phox translocation. Finally, treatment with ethanol increased both TNF-α production and neutrophil migration in CSM. The major new finding of this study is that NADPH oxidase-derived reactive oxygen species play a role on chronic ethanol consumption-induced ED and endothelial dysfunction in the rat CSM. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxidase catalysis via aerobically generated hypervalent iodine intermediates

NASA Astrophysics Data System (ADS)

Maity, Asim; Hyun, Sung-Min; Powers, David C.

2018-02-01

The development of sustainable oxidation chemistry demands strategies to harness O2 as a terminal oxidant. Oxidase catalysis, in which O2 serves as a chemical oxidant without necessitating incorporation of oxygen into reaction products, would allow diverse substrate functionalization chemistry to be coupled to O2 reduction. Direct O2 utilization suffers from intrinsic challenges imposed by the triplet ground state of O2 and the disparate electron inventories of four-electron O2 reduction and two-electron substrate oxidation. Here, we generate hypervalent iodine reagents—a broadly useful class of selective two-electron oxidants—from O2. This is achieved by intercepting reactive intermediates of aldehyde autoxidation to aerobically generate hypervalent iodine reagents for a broad array of substrate oxidation reactions. The use of aryl iodides as mediators of aerobic oxidation underpins an oxidase catalysis platform that couples substrate oxidation directly to O2 reduction. We anticipate that aerobically generated hypervalent iodine reagents will expand the scope of aerobic oxidation chemistry in chemical synthesis.

Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs.

PubMed

Endo, Daiki; Fujimoto, Kenta; Hirose, Rika; Yamanaka, Hiroko; Homme, Mizuki; Ishibashi, Ken-Ichi; Miura, Noriko; Ohno, Naohito; Aratani, Yasuaki

2017-02-01

Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases.

PubMed

Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

2014-08-01

The only known function of the Nox family of NADPH oxidases is the production of reactive oxygen species (ROS). Some Nox enzymes show high tissue-specific expression and the ROS locally produced are required for synthesis of hormones or tissue components. In the cardiovascular system, Nox enzymes are low abundant and function as redox-modulators. By reacting with thiols, nitric oxide (NO) or trace metals, Nox-derived ROS elicit a plethora of cellular responses required for physiological growth factor signaling and the induction and adaptation to pathological processes. The interactions of Nox-derived ROS with signaling elements in the cardiovascular system are highly diverse and will be detailed in this article, which is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System". Copyright © 2014 Elsevier Ltd. All rights reserved.

Following glucose oxidase activity by chemiluminescence and chemiluminescence resonance energy transfer (CRET) processes involving enzyme-DNAzyme conjugates.

PubMed

Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

2011-01-01

A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H(2)O(2). The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose.

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

PubMed

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.

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

PubMed

Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

2009-08-01

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

Following Glucose Oxidase Activity by Chemiluminescence and Chemiluminescence Resonance Energy Transfer (CRET) Processes Involving Enzyme-DNAzyme Conjugates

PubMed Central

Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

2011-01-01

A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H2O2. The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose. PMID:22346648

Xanthine Oxidase Induces Foam Cell Formation through LOX-1 and NLRP3 Activation.

PubMed

Dai, Yao; Cao, Yongxiang; Zhang, Zhigao; Vallurupalli, Srikanth; Mehta, Jawahar L

2017-02-01

-inflammatory mediators such as Caspase-1, IL-1β and IL-18. Xanthine oxidase also induces CD36 expression. Activation of both LOX-1 and CD36 (LOX-1> > CD36) participates in the transformation of macrophages and VSMCs into foam cells. Uric acid formed from xanthine-xanthine oxidase interaction stimulates CD36 expression and triggers foam cell formation independent of NLRP3 activation.

Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

PubMed Central

Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

2015-01-01

Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

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

PubMed

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.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

PubMed

Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

2013-12-15

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 on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

PubMed Central

Singh, Melissa M.; Irwin, Mary E.; Gao, Yin; Ban, Kechen; Shi, Ping; Arlinghaus, Ralph B.; Amin, Hesham M.; Chandra, Joya

2011-01-01

Background Patients with blast crisis phase chronic myelogeneous leukemia (CML) have poor response to tyrosine kinase inhibitors designed to inhibit the BCR-ABL1 oncogene. Recent work has shown that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1 expressing cells and that inhibition of HO-1 in CML leads to reduced cellular growth suggesting HO-1 may be a plausible target for therapy. Here we sought to clarify the mechanism of HO-1 overexpression and the role of the NADPH oxidase as a contributor to this mechanism in CML. Methods HO-1 expression was evaluated in CML bone marrow specimens from patients in various stages of disease, in a transplant based model for CML and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. Results Blast crisis CML patient specimens displayed higher levels of HO-1 staining than chronic or accelerated phase. HO-1 upregulation in BCR-ABL1 expressing cells was suppressed by diphenyliodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNAi to Rac1, a dominant negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed towards p47phox similarly abrogated HO-1 levels. Conclusion BCR-ABL1 expression upregulates HO-1, a survival factor for CML cells. This upregulation is more pronounced in blast crisis CML relative to early stage disease and is mediated by the NADPH oxidase components Rac1 and p47phox. Expression of p47phox is increased in BCR-ABL1 expressing cells. PMID:22139798

NADPH oxidase inhibitors: a patent review.

PubMed

Kim, Jung-Ae; Neupane, Ganesh Prasad; Lee, Eung Seok; Jeong, Byeong-Seon; Park, Byung Chul; Thapa, Pritam

2011-08-01

NADPH oxidases, a family of multi-subunit enzyme complexes, catalyze the production of reactive oxygen species (ROS), which may contribute to the pathogenesis of a variety of diseases. In addition to the first NADPH oxidase found in phagocytes, four non-phagocytic NADPH oxidase isoforms have been identified, which all differ in their catalytic subunit (Nox1-5) and tissue distribution. This paper provides a comprehensive review of the patent literature on NADPH oxidase inhibitors, small molecule Nox inhibitors, peptides and siRNAs. Since each member of the NADPH oxidase family has great potential as a therapeutic target, several different compounds have been registered as NADPH oxidase inhibitors in the patent literature. As yet, none have gone through clinical trials, and some have not completed preclinical trials, including safety and specificity evaluation. Recently, small molecule pyrazolopyridine and triazolopyrimidine derivatives have been submitted as potent NADPH oxidase inhibitors and reported as first-in-class inhibitors for idiopathic pulmonary fibrosis and acute stroke, respectively. Further clinical efficacy and safety data are warranted to prove their actual clinical utility.

SIRPα controls the activity of the phagocyte NADPH oxidase by restricting the expression of gp91(phox).

PubMed

van Beek, Ellen M; Zarate, Julian Alvarez; van Bruggen, Robin; Schornagel, Karin; Tool, Anton T J; Matozaki, Takashi; Kraal, Georg; Roos, Dirk; van den Berg, Timo K

2012-10-25

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as well as signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

PubMed Central

Basuroy, Shyamali; Tcheranova, Dilyara; Bhattacharya, Sujoy; Leffler, Charles W.

2011-01-01

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease. PMID:21123734

NADPH oxidase-mediated redox signal contributes to lipoteichoic acid-induced MMP-9 upregulation in brain astrocytes

PubMed Central

2012-01-01

Background Lipoteichoic acid (LTA) is a component of gram-positive bacterial cell walls and may be elevated in the cerebrospinal fluid of patients suffering from meningitis. Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Moreover, several studies have suggested that increased oxidative stress is implicated in the pathogenesis of brain inflammation and injury. However, the molecular mechanisms underlying LTA-induced redox signal and MMP-9 expression in brain astrocytes remain unclear. Objective Herein we explored whether LTA-induced MMP-9 expression was mediated through redox signals in rat brain astrocytes (RBA-1 cells). Methods Upregulation of MMP-9 by LTA was evaluated by zymographic and RT-PCR analyses. Next, the MMP-9 regulatory pathways were investigated by pretreatment with pharmacological inhibitors or transfection with small interfering RNAs (siRNAs), Western blotting, and chromatin immunoprecipitation (ChIP)-PCR and promoter activity reporter assays. Moreover, we determined the cell functional changes by migration assay. Results These results showed that LTA induced MMP-9 expression via a PKC(α)-dependent pathway. We further demonstrated that PKCα stimulated p47phox/NADPH oxidase 2 (Nox2)-dependent reactive oxygen species (ROS) generation and then activated the ATF2/AP-1 signals. The activated-ATF2 bound to the AP-1-binding site of MMP-9 promoter, and thereby turned on MMP-9 gene transcription. Additionally, the co-activator p300 also contributed to these responses. Functionally, LTA-induced MMP-9 expression enhanced astrocytic migration. Conclusion These results demonstrated that in RBA-1 cells, activation of ATF2/AP-1 by the PKC(α)-mediated Nox(2)/ROS signals is essential for upregulation of MMP-9 and cell migration enhanced by LTA. PMID:22643046

Substance P enhances microglial density in the substantia nigra through neurokinin-1 receptor/NADPH oxidase-mediated chemotaxis in mice.

PubMed

Wang, Qingshan; Oyarzabal, Esteban; Wilson, Belinda; Qian, Li; Hong, Jau-Shyong

2015-10-01

The distribution of microglia varies greatly throughout the brain. The substantia nigra (SN) contains the highest density of microglia among different brain regions. However, the mechanism underlying this uneven distribution remains unclear. Substance P (SP) is a potent proinflammatory neuropeptide with high concentrations in the SN. We recently demonstrated that SP can regulate nigral microglial activity. In the present study, we further investigated the involvement of SP in modulating nigral microglial density in postnatal developing mice. Nigral microglial density was quantified in wild-type (WT) and SP-deficient mice from postnatal day 1 (P1) to P30. SP was detected at high levels in the SN as early as P1 and microglial density did not peak until around P30 in WT mice. SP-deficient mice (TAC1(-/-)) had a significant reduction in nigral microglial density. No differences in the ability of microglia to proliferate were observed between TAC1(-/-) and WT mice, suggesting that SP may alter microglial density through chemotaxic recruitment. SP was confirmed to dose-dependently attract microglia using a trans-well culture system. Mechanistic studies revealed that both the SP receptor neurokinin-1 receptor (NK1R) and the superoxide-producing enzyme NADPH oxidase (NOX2) were necessary for SP-mediated chemotaxis in microglia. Furthermore, genetic ablation and pharmacological inhibition of NK1R or NOX2 attenuated SP-induced microglial migration. Finally, protein kinase Cδ (PKCδ) was recognized to couple SP/NK1R-mediated NOX2 activation. Altogether, we found that SP partly accounts for the increased density of microglia in the SN through chemotaxic recruitment via a novel NK1R-NOX2 axis-mediated pathway. © 2015 Authors; published by Portland Press Limited.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

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)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup 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

Periostin promotes liver fibrogenesis by activating lysyl oxidase in hepatic stellate cells.

PubMed

Kumar, Pradeep; Smith, Tekla; Raeman, Reben; Chopyk, Daniel M; Brink, Hannah; Liu, Yunshan; Sulchek, Todd; Anania, Frank A

2018-06-25

Liver fibrosis arises from dysregulated wound healing due to persistent inflammatory hepatic injury. Periostin is a non-structural extracellular matrix protein that promotes organ fibrosis in adults. Here, we sought to identify the molecular mechanisms in periostin-mediated hepatic fibrosis. Hepatic fibrosis in periostin -/- mice was attenuated as evidenced by significantly reduced collagen fibril density and liver stiffness compared with those in WT controls. A single dose of carbon tetrachloride caused similar acute liver injury in periostin -/- and WT littermates, and we did not detect significant differences in transaminases and major fibrosis-related hepatic gene expression between these two genotypes. Activated hepatic stellate cells (HSCs) are the major periostin-producing liver cell type. We found that in primary rat HSCs in vitro, periostin significantly increases the expression levels and activities of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) isoforms 1-3. Periostin also induced expression of intra- and extracellular collagen type 1 and fibronectin in HSCs. Interestingly, periostin stimulated phosphorylation of SMAD2/3, which was sustained despite sh-RNA mediated knockdown of transforming growth factor β (TGFβ) receptor I and II, indicating that periostin periostin-mediated SMAD2/3 phosphorylation is independent of TGFβ receptors. Moreover, periostin induced the phosphorylation of focal adhesion kinase (FAK) and AKT in HSCs. Notably, si-RNA mediated FAK knockdown failed to block periostin-induced SMAD2/3 phosphorylation. These results suggest that periostin promotes enhanced matrix stiffness in chronic liver disease by activating LOX and LOXL, independently of TGFβ receptors. Hence, targeting periostin may be of therapeutic benefit in combating hepatic fibrosis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

He, Yan; Cui, Jiankun; Lee, James C-M; Ding, Shinghua; Chalimoniuk, Malgorzata; Simonyi, Agnes; Sun, Albert Y; Gu, Zezong; Weisman∥, Gary A; Gibson Wood, W; Sun, Grace Y

2011-01-01

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 pre-treatment 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. PMID:21434871

Targeting NADPH oxidases in vascular pharmacology

PubMed Central

Schramm, Agata; Matusik, Paweł; Osmenda, Grzegorz; Guzik, Tomasz J

2012-01-01

Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the

Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

PubMed

Spiekermann, Stephan; Landmesser, Ulf; Dikalov, Sergey; Bredt, Martin; Gamez, Graciela; Tatge, Helma; Reepschläger, Nina; Hornig, Burkhard; Drexler, Helmut; Harrison, David G

2003-03-18

Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

Isolated sulfite oxidase deficiency.

PubMed

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.

NADPH Oxidases in Vascular Pathology

PubMed Central

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

2014-01-01

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

Aspects on mediated glucose oxidation at a supported cubic phase.

PubMed

Aghbolagh, Mahdi Shahmohammadi; Khani Meynaq, Mohammad Yaser; Shimizu, Kenichi; Lindholm-Sethson, Britta

2017-12-01

A supported liquid crystalline cubic phase housing glucose oxidase on an electrode surface has been suggested as bio-anode in a biofuel. The purpose of this investigation is to clarify some aspect on the mediated enzymatic oxidation of glucose in such a bio-anode where the mediator ferrocene-carboxylic acid and glucose were dissolved in the solution. The enzyme glucose oxidase was housed in the water channels of the mono-olein cubic phase. The system was investigated with cyclic voltammetry at different scan rates and the temperature was varied between 15°C and 30°C. The diffusion coefficient of the mediator and also the film resistance was estimated showing a large decrease in the mass-transport properties as the temperature was decreased. The current from mediated oxidation of glucose at the electrode surface increased with decreasing film thickness. The transport of the mediator in the cubic phase was the rate-limiting step in the overall reaction, where the oxidation of glucose took place at the outer surface of the cubic phase. Copyright © 2017 Elsevier B.V. All rights reserved.

Vasoactive intestinal peptide prevents lung injury due to xanthine/xanthine oxidase.

PubMed

Berisha, H; Foda, H; Sakakibara, H; Trotz, M; Pakbaz, H; Said, S I

1990-08-01

Reactive oxygen species mediate injury and inflammation in many tissues. The addition of xanthine and xanthine oxidase to perfused rat lungs led to increases in peak airway pressure and perfusion pressure, pulmonary edema, and increased protein content in bronchoalveolar lavage fluid. Treatment with 1-10 micrograms.kg-1.min-1 of vasoactive intestinal peptide (VIP), a widely distributed neuropeptide, markedly reduced or totally prevented all signs of injury. Simultaneously, VIP also diminished or abolished the associated generation of arachidonate products. Similar protection was provided by catalase (100 micrograms/ml) but not by the VIP-related peptides secretin or glucagon. The pulmonary vasodilator papaverine (0.15 mg/ml) was also ineffective. Injured lungs that were not treated with VIP released large amounts of this peptide in the perfusate. The results indicate that VIP has potent protective activity against injury triggered by xanthine/xanthine oxidase and may be a physiological modulator of inflammatory tissue damage associated with toxic oxygen metabolites.

A mediator-free glucose biosensor based on glucose oxidase/chitosan/α-zirconium phosphate ternary biocomposite.

PubMed

Liu, Li-Min; Wen, Jiwu; Liu, Lijun; He, Deyong; Kuang, Ren-yun; Shi, Taqing

2014-01-15

A novel glucose oxidase/chitosan/α-zirconium phosphate (GOD/chitosan/α-ZrP) ternary biocomposite was prepared by co-intercalating glucose oxidase (GOD) and chitosan into the interlayers of α-zirconium phosphate (α-ZrP) via a delamination-reassembly procedure. The results of X-ray diffraction, infrared spectroscopy, circular dichroism, and ultraviolet spectrum characterizations indicated not only the layered and hybrid structure of the GOD/chitosan/α-ZrP ternary biocomposite but also the recovered activity of the intercalated GOD improved by the co-intercalated chitosan. By depositing the GOD/chitosan/α-ZrP biocomposite film onto a glassy carbon electrode, the direct electrochemistry of the intercalated GOD was achieved with a fast electron transfer rate constant, k(s), of 7.48±3.52 s(-1). Moreover, this GOD/chitosan/α-ZrP biocomposite modified electrode exhibited a sensitive response to glucose in the linear range of 0.25-8.0 mM (R=0.9994, n=14), with a determination limit of 0.076 mM. Copyright © 2013 Elsevier Inc. All rights reserved.

SPERMINE OXIDASE: AN AMINE OXIDASE WITH SPECIFICITY FOR SPERMINE AND SPERMIDINE

PubMed Central

Hirsch, James G.

1953-01-01

Sheep serum and bovine serum contain an enzyme which brings about a rapid oxidative deamination of certain biological amines. This enzyme differs from previously described amine oxidases in several regards and especially in its substrate specificity. Studies thus far indicate that only spermine and the closely related compound spermidine serve as substrates for the enzyme in sheep serum. For this reason, the enzyme has been named spermine oxidase. Spermine oxidase is active in a variety of fluids of various ionic strength and buffer composition. The reaction takes place between pH 6.0 and pH 8.0 with an optimal rate in the vicinity of neutrality. Under certain conditions, the rate of oxygen consumption during the initial phase of the reaction is independent of the concentration of substrate. The diminution in rate observed during the latter phase of the enzymatic attack appears to be due to an alteration in the kinetics at low concentrations of substrate, or to competitive inhibition by a product of the reaction. Carbonyl reagents almost completely block the action of spermine oxidase, while certain amines and the cyanide ion bring about partial inhibition. Thiol reagents and sequestering compounds do not alter the course of the oxidative process. In the presence of low concentrations of mercuric chloride, the sheep serum-spermine system consumes approximately twice as much oxygen as controls containing no mercuric ion. The mechanism by which the mercuric ion stimulates additional oxygen uptake is obscure. PMID:13052805

A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase

PubMed Central

Venema, K.; van Berkel, W. J. H.; Korf, J.

2007-01-01

This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested. Figure Preparation of apoGOx and restoration of enzyme activity using a soluton of FAD PMID:17909761

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes.

PubMed

Bour, Sandy; Daviaud, Danièle; Gres, Sandra; Lefort, Corinne; Prévot, Danielle; Zorzano, Antonio; Wabitsch, Martin; Saulnier-Blache, Jean-Sébastien; Valet, Philippe; Carpéné, Christian

2007-08-01

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix.

PubMed

Cil, M; Böyükbayram, A E; Kiralp, S; Toppare, L; Yağci, Y

2007-06-01

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-α in cerebral vascular endothelial cells

PubMed Central

Basuroy, Shyamali; Bhattacharya, Sujoy; Leffler, Charles W.; Parfenova, Helena

2009-01-01

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-α causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (Nω-nitro-l-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-α-induced superoxide (O2•−) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-α-evoked O2•− generation and apoptosis. TNF-α rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O2•− production, and accounts for a burst of oxidative stress in response to TNF-α. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-α in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF-α toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O2•− and apoptosis caused by TNF-α stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-α-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-α-induced oxidative stress by inhibiting Nox4 activity and/or by O2•− scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease. PMID:19118162

Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.

PubMed Central

Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi

2004-01-01

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase. PMID:14594451

VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

PubMed

Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

2016-08-01

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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

PubMed

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.

The Role of Aldehyde Oxidase and Xanthine Oxidase in the Biotransformation of a Novel Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 5

PubMed Central

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

A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

PubMed

Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

2014-01-01

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

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

PubMed

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.

Promising perspectives for ruminal protection of polyunsaturated fatty acids through polyphenol-oxidase-mediated crosslinking of interfacial protein in emulsions.

PubMed

De Neve, N; Vlaeminck, B; Gadeyne, F; Claeys, E; Van der Meeren, P; Fievez, V

2018-03-16

Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions (<50

Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

PubMed Central

Miyata, Naoyuki; Tani, Yukinori; Maruo, Kanako; Tsuno, Hiroshi; Sakata, Masahiro; Iwahori, Keisuke

2006-01-01

Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes. PMID:17021194

The interaction between monoamine oxidase A and punitive discipline in the development of antisocial behavior: Mediation by maladaptive social information processing.

PubMed

Galán, Chardée A; Choe, Daniel Ewon; Forbes, Erika E; Shaw, Daniel S

2017-10-01

Previous studies demonstrate that boys' monoamine oxidase A (MAOA) genotype interacts with adverse rearing environments in early childhood, including punitive discipline, to predict later antisocial behavior. Yet the mechanisms by which MAOA and punitive parenting interact during childhood to amplify risk for antisocial behavior are not well understood. In the present study, hostile attributional bias and aggressive response generation during middle childhood, salient aspects of maladaptive social information processing, were tested as possible mediators of this relation in a sample of 187 low-income men followed prospectively from infancy into early adulthood. Given racial-ethnic variation in MAOA allele frequencies, analyses were conducted separately by race. In both African American and Caucasian men, those with the low-activity MAOA allele who experienced more punitive discipline at age 1.5 generated more aggressive responses to perceived threat at age 10 relative to men with the high-activity variant. In the African American subsample only, formal mediation analyses indicated a marginally significant indirect effect of maternal punitiveness on adult arrest records via aggressive response generation in middle childhood. The findings suggest that maladaptive social information processing may be an important mechanism underlying the association between MAOA × Parenting interactions and antisocial behavior in early adulthood. The present study extends previous work in the field by demonstrating that MAOA and harsh parenting assessed in early childhood interact to not only predict antisocial behavior in early adulthood, but also predict social information processing, a well-established social-cognitive correlate of antisocial behavior.

Correlation Between Monoamine Oxidase Inhibitors and Anticonvulsants

PubMed Central

Dwivedi, Chandradhar; Misra, Radhey S.; Chaudhari, Anshumali; Parmar, Surendra S.

1980-01-01

Monoamine oxidase inhibitory and anticonvulsant properties of 2-substituted styryl-6-bromo-3-(4-ethylbenzoate/4 benzhydrazide)-4-quinazoles are studied. All styryl quinazolone esters except compound number 9 exhibited monoamine oxidase inhibitory properties during oxidative deamination of kynuramine. Corresponding hydrazides were found to have relatively higher activity. All these quinazolones were able to protect against pentylenetetrazol induced seizures. These observations in general do not prove that monoamine oxidase inhibitory properties represent the biochemical basis for the anticonvulsant activity of these compounds. PMID:7420438

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

PubMed Central

Okamura, H; Murooka, Y; Harada, T

1976-01-01

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation. PMID:179974

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

PubMed

Shepherd, Mark; Achard, Maud E S; Idris, Adi; Totsika, Makrina; Phan, Minh-Duy; Peters, Kate M; Sarkar, Sohinee; Ribeiro, Cláudia A; Holyoake, Louise V; Ladakis, Dimitrios; Ulett, Glen C; Sweet, Matthew J; Poole, Robert K; McEwan, Alastair G; Schembri, Mark A

2016-10-21

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease

PubMed Central

Pushkaran, Suvarnamala; Konstantinidis, Diamantis G.; Koochaki, Sebastian; Malik, Punam; Mohandas, Narla; Zheng, Yi; Joiner, Clinton H.; Kalfa, Theodosia A.

2013-01-01

Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca2+ signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor β1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD. PMID:23349388

NADPH oxidases of the brain: distribution, regulation, and function.

PubMed

Infanger, David W; Sharma, Ram V; Davisson, Robin L

2006-01-01

The NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of molecular oxygen to form superoxide (O(2)(-)). While classically linked to the respiratory burst in neutrophils, recent evidence now shows that O(2)(-) (and associated reactive oxygen species, ROS) generated by NADPH oxidase in nonphagocytic cells serves myriad functions in health and disease. An entire new family of NADPH Oxidase (Nox) homologues has emerged, which vary widely in cell and tissue distribution, as well as in function and regulation. A major concept in redox signaling is that while NADPH oxidase-derived ROS are necessary for normal cellular function, excessive oxidative stress can contribute to pathological disease. This certainly is true in the central nervous system (CNS), where normal NADPH oxidase function appears to be required for processes such as neuronal signaling, memory, and central cardiovascular homeostasis, but overproduction of ROS contributes to neurotoxicity, neurodegeneration, and cardiovascular diseases. Despite implications of NADPH oxidase in normal and pathological CNS processes, still relatively little is known about the mechanisms involved. This paper summarizes the evidence for NADPH oxidase distribution, regulation, and function in the CNS, emphasizing the diversity of Nox isoforms and their new and emerging role in neuro-cardiovascular function. In addition, perspectives for future research and novel therapeutic targets are offered.

Gravity Responsive NADH Oxidase of the Plasma Membrane

NASA Technical Reports Server (NTRS)

Morre, D. James (Inventor)

2002-01-01

A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

Immobilization of Pichia pastoris cells containing alcohol oxidase activity

PubMed Central

Maleknia, S; Ahmadi, H; Norouzian, D

2011-01-01

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

Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

PubMed Central

van den Heuvel, Robert H. H.; Fraaije, Marco W.; Laane, Colja; van Berkel, Willem J. H.

1998-01-01

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (R)-1-(4′-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171–177, 1998). (S)-1-(4′-Hydroxyphenyl)ethanol was found to be a far better substrate than (R)-1-(4′-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4′-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4′-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols. PMID:9791114

Novel ferrocene-anchored ZnO nanoparticle/carbon nanotube assembly for glucose oxidase wiring: application to a glucose/air fuel cell.

PubMed

Haddad, Raoudha; Mattei, Jean-Gabriel; Thery, Jessica; Auger, Aurélien

2015-06-28

Glucose oxidase (GOx) is immobilized on ZnO nanoparticle-modified electrodes. The immobilized glucose oxidase shows efficient mediated electron transfer with ZnO nanoparticles to which the ferrocenyl moiety is π-stacked into a supramolecular architecture. The constructed ZnO-Fc/CNT modified electrode exhibits high ferrocene surface coverage, preventing any leakage of the π-stacked ferrocene from the newly described ZnO hybrid nanoparticles. The use of the new architecture of ZnO supported electron mediators to shuttle electrons from the redox centre of the enzyme to the surface of the working electrode can effectively bring about successful glucose oxidation. These modified electrodes evaluated as a highly efficient architecture provide a catalytic current for glucose oxidation and are integrated in a specially designed glucose/air fuel cell prototype using a conventional platinum-carbon (Pt/C) cathode at physiological pH (7.0). The obtained architecture leads to a peak power density of 53 μW cm(-2) at 300 mV for the Nafion® based biofuel cell under "air breathing" conditions at room temperature.

Multicopper Oxidase Involvement in Both Mn(II) and Mn(III) Oxidation during Bacterial Formation of MnO2

PubMed Central

Soldatova, Alexandra V.; Butterfield, Cristina; Oyerinde, Oyeyemi F.; Tebo, Bradley M.; Spiro, Thomas G.

2013-01-01

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO2 formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria implicates multicopper oxidases (MCOs) as being required for MnO2 formation. However, MCOs catalyze one-electron oxidations, whereas conversion of Mn(II) to MnO2 is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O2 and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO2 also depends on O2 and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO2 formation. PMID:22892957

Radical-Mediated Enzymatic Polymerizations

PubMed Central

Zavada, Scott R.; Battsengel, Tsatsral; Scott, Timothy F.

2016-01-01

Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes―catalytic proteins―owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol–ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. PMID:26848652

Clinical potential of phycocyanobilin for induction of T regulatory cells in the management of inflammatory disorders.

PubMed

McCarty, Mark F

2011-12-01

Exposure of human mononuclear cells to phycocyanin in vitro is reported to promote generation of Treg cells. Induction of heme oxygenase-1 (HO-1) in lymphocytes has a similar effect, and it is not likely to be accidental that a key product of HO-1 activity, biliverdin, is homologous to the structure of phycocyanin's chromophore phycocyanobilin (PhyCB). Moreover, Treg induction is observed in mice injected with bilirubin, biliverdin's chief metabolite. These considerations suggest that bilirubin, generated within lymphocytes by HO-1 activation, may play a physiological role in the promotion of Treg immunomodulation. This effect of bilirubin is likely to be independent of NADPH oxidase inhibition, since the NAPDH oxidase activity of macrophages is necessary for Treg induction, possibly because it contributes to HO-1 induction in lymphocytes. In light of numerous reports that oral phycocyanin is beneficial in various rodent models of autoimmune disorders, it is reasonable to suspect that PhyCB-enriched spirulina extracts may have clinical potential for boosting Treg activity in human autoimmune or allergic syndromes, mimicking the physiological role of HO-1 induction in this regard. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

PubMed Central

Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

2012-01-01

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

Development of mediator-type biosensor to wirelessly monitor whole cholesterol concentration in fish.

PubMed

Takase, Mai; Murata, Masataka; Hibi, Kyoko; Huifeng, Ren; Endo, Hideaki

2014-04-01

We developed a wireless monitoring system to monitor fish condition by tracking the change in whole cholesterol concentration. The whole cholesterol concentration of fish is a source of steroid hormones or indicator of immunity level, which makes its detection important for tracking physiological condition of fish. Wireless monitoring system comprises of mediator-type biosensor and wireless transmission device. Biosensor is implantable to fish body, and transmission device is so light, in that fish is allowed to swim freely during monitoring. Cholesterol esterase and oxidase were fixated on to the detection site of biosensor and used to detect the whole cholesterol concentration. However, cholesterol oxidase incorporates oxidation-reduction reaction of oxygen for detection, which concentration fluctuates easily due to change in environmental condition. Meanwhile, mediator-type biosensor enables monitoring of whole cholesterol concentration by using mediator to substitute that oxidation-reduction reaction of oxygen. Characteristic of fabricated mediator-type biosensor was tested. The sensor output current of mediator-type biosensor remained stable compared to output current of non-mediator-type biosensor under fluctuating oxygen concentration of 0-8 ppm, which implied that this sensor is less affected by change in dissolved oxygen concentration. That biosensor was then implanted into fish for wireless monitoring. As a result, approximately 48 h of real-time monitoring was successful.

Crystal Structure of Alcohol Oxidase from Pichia pastoris

PubMed Central

Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

2016-01-01

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

Calcium transport in vesicles energized by cytochrome oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosier, Randy N.

1979-01-01

Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K + selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K + flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interactionmore » with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.« less

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

PubMed

Cervelli, Manuela; Polticelli, Fabio; Angelucci, Emanuela; Di Muzio, Elena; Stano, Pasquale; Mariottini, Paolo

2015-05-01

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

PubMed

Castresana, J; Lübben, M; Saraste, M; Higgins, D G

1994-06-01

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

Inhibition of Human Vascular NADPH Oxidase by Apocynin Derived Oligophenols

PubMed Central

Mora-Pale, Mauricio; Weïwer, Michel; Yu, Jingjing; Linhardt, Robert J.; Dordick, Jonathan S.

2009-01-01

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47phox and p22phox. To that end, while apocynin was unable to block the interaction of his-tagged p47phox with a surface immobilized biotinalyted p22phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase. PMID:19523836

Current status of NADPH oxidase research in cardiovascular pharmacology.

PubMed

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

2013-01-01

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

Current status of NADPH oxidase research in cardiovascular pharmacology

PubMed Central

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

2013-01-01

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

Oxygen activation in flavoprotein oxidases: the importance of being positive.

PubMed

Gadda, Giovanni

2012-04-03

The oxidation of flavin hydroquinones by O(2) in solution is slow, with second-order rate constants of ~250 M(-1) s(-1). This is due to the obligatory, single-electron transfer that initiates the reaction being thermodynamically unfavored and poorly catalyzed. Notwithstanding considerations of O(2) accessibility to the reaction site, its desolvation and geometry and other factors that can also contribute to further rate acceleration, flavoprotein oxidases must activate O(2) for reaction with flavin hydroquinones to be able to achieve the 100-1000-fold rate enhancements typically observed. Protein positive charges have been identified in glucose oxidase, monomeric sarcosine oxidase, N-methyltryptophan oxidase and fructosamine oxidase that electrostatically stabilize the transition state for the initial single electron transfer that generates the O(2)(-•)/flavin semiquinone radical pair. In choline oxidase despite the presence of three histidines in the active site, the trimethylammonium group of the reaction product provides such an electrostatic stabilization. A nonpolar site proximal to the flavin C(4a) atom in choline oxidase has also been identified, which contributes to the geometry and desolvation of the O(2) reaction site. The relevance of O(2) activation by product charges to other flavoprotein oxidases, such as for example those catalyzing amine oxidations, is discussed in this review. A nonpolar site close to the flavin C(4a) atom and a positive charge is identified through structural analysis in several flavoprotein oxidases. Mutagenesis has disclosed nonpolar sites in O(2)-reducing enzymes that utilize copper/TPQ or iron. It is predicted that classes of O(2)-reducing enzymes utilizing other cofactors also contain a similar catalytic motif.

Alcohol oxidase protein mediated in-situ synthesized and stabilized gold nanoparticles for developing amperometric alcohol biosensor.

PubMed

Chinnadayyala, Somasekhar R; Santhosh, Mallesh; Singh, Naveen K; Goswami, Pranab

2015-07-15

A simple one step method for the alcohol oxidases (AOx) protein mediated synthesis of gold nano-particles (AuNPs) in alkaline (pH 8.5) condition with simultaneous stabilization of the nanoparticles on the AOx protein surface under native environment has been developed. The formation of the AOx conjugated AuNPs was confirmed by advanced analytical and spectroscopic techniques. The significant increase in zeta potential (ζ) value of -57mV for the synthesized AOx-AuNPs conjugate from the AOx (pI 4.5) protein (ζ, -30mV) implied good stability of the in-situ synthesized nano-conjugate. The AOx-AuNPs conjugate showed steady stability in alkaline (upto pH 8.5) and NaCl (up to 10(-1)M) solutions. The efficiency (Kcat/Km) of the AuNP conjugated AOx was increased by 18% from the free enzyme confirming the activating role of the surface stabilized AuNPs for the enzyme. The AuNPs-AOx conjugate was encapsulated with polyaniline (PANI) synthesized by oxidative polymerization of aniline using H2O2 generated in-situ from the AOx catalysed oxidation of alcohol. The PANI encapsulated AuNPs-AOx assembly was stabilized on a glassy carbon electrode (GCE) by chitosan-Nafion mixture and then utilized the fabricated bioelectrode for detection of alcohol amperometrically using H2O2 as redox indicator at +0.6V. The constructed biosensor showed high operational stability (6.3% loss after 25 measurements), wide linear detection range of 10µM-4.7mM (R(2)=0.9731), high sensitivity of 68.3±0.35µAmM(-1) and low detection limit of 7±0.027µM for ethanol. The fabricated bioelectrode was successfully used for the selective determination of alcohol in beverage samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Graphene oxide-mediated electrochemistry of glucose oxidase on glassy carbon electrodes.

PubMed

Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco; Sadeghi, Sheila J

2016-01-01

Glucose oxidase (GOD) was immobilized on glassy carbon electrodes in the presence of graphene oxide (GO) as a model system for the interaction between GO and biological molecules. Lyotropic properties of didodecyldimethylammonium bromide (DDAB) were used to stabilize the enzymatic layer on the electrode surface resulting in a markedly improved electrochemical response of the immobilized GOD. Transmission electron microscopy images of the GO with DDAB confirmed the distribution of the GO in a two-dimensional manner as a foil-like material. Although it is known that glassy carbon surfaces are not ideal for hydrogen peroxide detection, successful chronoamperometric titrations of the GOD in the presence of GO with β-d-glucose were performed on glassy carbon electrodes, whereas no current response was detected upon β-d-glucose addition in the absence of GO. The GOD-DDAB-GO system displayed a high turnover efficiency and substrate affinity as a glucose biosensor. The simplicity and ease of the electrode preparation procedure of this GO/DDAB system make it a good candidate for immobilizing other biomolecules for fabrication of amperometric biosensors. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

The First Mammalian Aldehyde Oxidase Crystal Structure

PubMed Central

Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

2012-01-01

Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

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

PubMed Central

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

2013-01-01

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

Generating disulfides with the quiescin sulfhydryl oxidases

PubMed Central

Heckler, Erin J.; Rancy, Pumtiwitt C.; Kodali, Vamsi K.; Thorpe, Colin

2008-01-01

The Quiescin-sulfhydryl oxidase (QSOX) family of flavoenzymes catalyzes the direct and facile insertion of disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen to hydrogen peroxide. This review discusses the chemical mechanism of these enzymes and the involvement of thioredoxin and flavin-binding domains in catalysis. The variability of CxxC motifs in the QSOX family is highlighted and attention is drawn to the steric factors that may promote efficient thiol/disulfide exchange during oxidative protein folding. The varied cellular location of these multi-domain sulfhydryl oxidases is reviewed and potential intracellular and extracellular roles are summarized. Finally, this review identifies important unresolved questions concerning this ancient family of sulfhydryl oxidases. PMID:17980160

Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLCγ2 signaling axis in mice

PubMed Central

Graham, Daniel B.; Robertson, Charles M.; Bautista, Jhoanne; Mascarenhas, Francesca; Diacovo, M. Julia; Montgrain, Vivianne; Lam, Siu Kit; Cremasco, Viviana; Dunne, W. Michael; Faccio, Roberta; Coopersmith, Craig M.; Swat, Wojciech

2007-01-01

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C–γ2 (PLC-γ2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-γ2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav. PMID:17932569

Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: role of xanthine-oxidase and extracellular superoxide dismutase.

PubMed

Landmesser, Ulf; Spiekermann, Stephan; Dikalov, Sergey; Tatge, Helma; Wilke, Ragna; Kohler, Christoph; Harrison, David G; Hornig, Burkhard; Drexler, Helmut

2002-12-10

Impaired flow-dependent, endothelium-mediated vasodilation (FDD) in patients with chronic heart failure (CHF) results, at least in part, from accelerated degradation of nitric oxide by oxygen radicals. The mechanisms leading to increased vascular radical formation, however, remain unclear. Therefore, we determined endothelium-bound activities of extracellular superoxide dismutase (ecSOD), a major vascular antioxidant enzyme, and xanthine-oxidase, a potent radical producing enzyme, and their relation to FDD in patients with CHF. ecSOD and xanthine-oxidase activities, released from endothelium into plasma by heparin bolus injection, were determined in 14 patients with CHF and 10 control subjects. FDD of the radial artery was measured using high-resolution ultrasound and was assessed before and after administration of the antioxidant vitamin C (25 mg/min; IA). In patients with CHF, endothelium-bound ecSOD activity was substantially reduced (5.0+/-0.7 versus 14.4+/-2.6 U x mL(-1) x min(-1); P<0.01) and closely related to FDD (r=0.61). Endothelium-bound xanthine-oxidase activity was increased by >200% (38+/-10 versus 12+/-4 nmol O2*- x microL(-1); P<0.05) and inversely related to FDD (r=-0.35) in patients with CHF. In patients with low ecSOD and high xanthine-oxidase activity, a greater benefit of vitamin C on FDD was observed, ie, the portion of FDD inhibited by radicals correlated negatively with ecSOD (r=-0.71) but positively with xanthine-oxidase (r=0.75). These results demonstrate that both increased xanthine-oxidase and reduced ecSOD activity are closely associated with increased vascular oxidative stress in patients with CHF. This loss of vascular oxidative balance likely represents a novel mechanism contributing to endothelial dysfunction in CHF.

THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

PubMed Central

Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

1944-01-01

1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

PubMed

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.

Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

PubMed Central

Pick, Edgar

2014-01-01

The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

PubMed

Condino-Neto, A; Whitney, C; Newburger, P E

1998-11-01

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp06009a Click here for additional data file.

PubMed Central

Brugger, Dagmar; Sützl, Leander; Zahma, Kawah; Haltrich, Dietmar; Peterbauer, Clemens K.

2016-01-01

This study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold-nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active equivalent dehydrogenase via a single residue exchange. PMID:27808302

Heterologous expression and characterization of mouse spermine oxidase.

PubMed

Cervelli, Manuela; Polticelli, Fabio; Federico, Rodolfo; Mariottini, Paolo

2003-02-14

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production.

PubMed

Tamás, L; Budíková, S; Huttová, J; Mistrík, I; Simonovicová, M; Siroká, B

2005-06-01

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H(2)O(2) generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H(2)O(2). Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

PubMed

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.

In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

PubMed

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.

R1, a novel repressor of the human monoamine oxidase A.

PubMed

Chen, Kevin; Ou, Xiao-Ming; Chen, Gao; Choi, Si Ho; Shih, Jean C

2005-03-25

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.

A novel proteolytic processing of prolysyl oxidase

PubMed Central

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E.; Yamauchi, Mitsuo

2012-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residue Gly162 and Asp163 (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity and mass spectrometry. One form was identified as a well characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX (tLOX) resulting from the cleavage at the carboxy terminus of Arg192. The tLOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX. PMID:21591931

A novel proteolytic processing of prolysyl oxidase.

PubMed

Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Tokutomi, Kentaro; Mocanu, Viorel; Parker, Carol E; Yamauchi, Mitsuo

2011-01-01

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.

Exploiting algal NADPH oxidase for biophotovoltaic energy

DOE PAGES

Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

2015-01-29

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of type-2-copper-depleted ascorbate oxidase.

PubMed

O'Neill, P; Fielden, E M; Avigliano, L; Marcozzi, G; Ballini, A; Agrò, F

1984-08-15

The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.

Differential dose- and time-dependent effects of molindone on dopamine neurons of rat brain: mediation by irreversible inhibition of monoamine oxidase.

PubMed

Meller, E; Friedman, E

1982-03-01

The effects of molindone (2.5, 10 and 40 mg/kg) on striatal tyrosine hydroxylase activity and dopamine (DA), 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were measured as a function of time (0-72 hr). Whereas a dose of 2.5 mg/kg produced effects typical of DA receptor blockade (activation of synaptosomal tyrosine hydroxylase, increased DA metabolite levels and unchanged DA levels), a dose of 40 mg/kg produced opposite effects (decreased tyrosine hydroxylase activity and metabolite concentrations and elevated DA levels). A dose of 10 mg/kg elicited intermediate effects. The atypical effects of both higher doses were long-lasting (less than 72 hr). Molindone at doses of 10 or 40 mg/kg, but nor 2.5 mg/kg, selectively, irreversibly and dose-dependently inhibited type A monoamine oxidase. This inhibition appeared to be due to a metabolite, inasmuch as the drug itself inhibited monoamine oxidase (reversibly) only at high concentrations (less than or equal to 10(-4) M). The heretofore unsuspected inhibition of monoamine oxidase by molindone provided a consistent mechanistic interpretation of the differential dose- and time-dependent effects of the drug on dopaminergic neuronal activity. This mechanism may also serve to explain the reported efficacy of molindone in animal tests for antidepressant activity as well as its inability to produce increased DA receptor binding after chronic treatment.

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.

PubMed

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.

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina.

PubMed

Krause, Frank; Scheckhuber, Christian Q; Werner, Alexandra; Rexroth, Sascha; Reifschneider, Nicole H; Dencher, Norbert A; Osiewacz, Heinz D

2004-06-18

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

PubMed

Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

2013-04-01

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

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

PubMed

Shao, Beili; Bayraktutan, Ulvi

2014-01-01

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

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

PubMed Central

Shao, Beili; Bayraktutan, Ulvi

2014-01-01

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

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase.

PubMed

Rashidi, Mohammad-Reza; Beedham, Christine; Smith, John S; Davaran, Soodabeh

2007-08-01

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

PubMed

Ferrari, Alessandro R; Rozeboom, Henriëtte J; Dobruchowska, Justyna M; van Leeuwen, Sander S; Vugts, Aniek S C; Koetsier, Martijn J; Visser, Jaap; Fraaije, Marco W

2016-11-04

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

PubMed

Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

2016-10-01

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5.

PubMed

Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

2014-01-01

POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5.

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

PubMed Central

Molitor, Christian; Mauracher, Stephan Gerhard

2016-01-01

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

PubMed Central

Kallio, A; Jänne, J

1983-01-01

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone. PMID:6411077

Three-dimensional organization of three-domain copper oxidases: A review

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Zhukova, Yu. N.; Lyashenko, A. V.; Zaĭtsev, V. N.; Mikhaĭlov, A. M.

2008-01-01

“Blue” copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrena maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.

NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

PubMed

Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

2016-01-01

NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

PubMed Central

Kinnersley, Alan M.; Davies, Peter J.

1976-01-01

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

PubMed Central

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

Monoamine Oxidase A Gene Methylation and Its Role in Posttraumatic Stress Disorder: First Evidence from the South Eastern Europe (SEE)-PTSD Study

PubMed Central

Ziegler, Christiane; Wolf, Christiane; Schiele, Miriam A; Feric Bojic, Elma; Kucukalic, Sabina; Sabic Dzananovic, Emina; Goci Uka, Aferdita; Hoxha, Blerina; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend; Kravic, Nermina; Muminovic Umihanic, Mirnesa; Cima Franc, Ana; Jaksic, Nenad; Babic, Romana; Pavlovic, Marko; Warrings, Bodo; Bravo Mehmedbasic, Alma; Rudan, Dusko; Aukst-Margetic, Branka; Kucukalic, Abdulah; Marjanovic, Damir; Babic, Dragan; Bozina, Nada; Jakovljevic, Miro; Sinanovic, Osman; Avdibegovic, Esmina; Agani, Ferid; Dzubur-Kulenovic, Alma; Deckert, Jürgen; Domschke, Katharina

2018-01-01

Abstract Background Posttraumatic stress disorder is characterized by an overactive noradrenergic system conferring core posttraumatic stress disorder symptoms such as hyperarousal and reexperiencing. Monoamine oxidase A is one of the key enzymes mediating the turnover of noradrenaline. Here, DNA methylation of the monoamine oxidase A gene exonI/intronI region was investigated for the first time regarding its role in posttraumatic stress disorder risk and severity. Methods Monoamine oxidase A methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells in a total sample of N=652 (441 male) patients with current posttraumatic stress disorder, patients with remitted posttraumatic stress disorder, and healthy probands (comparison group) recruited at 5 centers in Bosnia-Herzegovina, Croatia, and the Republic of Kosovo. Posttraumatic stress disorder severity was measured by means of the Clinician-Administered Posttraumatic Stress Disorder Scale and its respective subscores representing distinct symptom clusters. Results In the male, but not the female sample, patients with current posttraumatic stress disorder displayed hypermethylation of 3 CpGs (CpG3=43656362; CpG12=43656514; CpG13=43656553, GRCh38.p2 Assembly) as compared with remitted Posttraumatic Stress Disorder patients and healthy probands. Symptom severity (Clinician-Administered Posttraumatic Stress Disorder Scale scores) in male patients with current posttraumatic stress disorder significantly correlated with monoamine oxidase A methylation. This applied particularly to symptom clusters related to reexperiencing of trauma (cluster B) and hyperarousal (cluster D). Conclusions The present findings suggest monoamine oxidase A gene hypermethylation, potentially resulting in enhanced noradrenergic signalling, as a disease status and severity marker of current posttraumatic stress disorder in males. If replicated, monoamine oxidase A hypermethylation might serve as a

Monoamine Oxidase A Gene Methylation and Its Role in Posttraumatic Stress Disorder: First Evidence from the South Eastern Europe (SEE)-PTSD Study.

PubMed

Ziegler, Christiane; Wolf, Christiane; Schiele, Miriam A; Feric Bojic, Elma; Kucukalic, Sabina; Sabic Dzananovic, Emina; Goci Uka, Aferdita; Hoxha, Blerina; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend; Kravic, Nermina; Muminovic Umihanic, Mirnesa; Cima Franc, Ana; Jaksic, Nenad; Babic, Romana; Pavlovic, Marko; Warrings, Bodo; Bravo Mehmedbasic, Alma; Rudan, Dusko; Aukst-Margetic, Branka; Kucukalic, Abdulah; Marjanovic, Damir; Babic, Dragan; Bozina, Nada; Jakovljevic, Miro; Sinanovic, Osman; Avdibegovic, Esmina; Agani, Ferid; Dzubur-Kulenovic, Alma; Deckert, Jürgen; Domschke, Katharina

2018-05-01

Posttraumatic stress disorder is characterized by an overactive noradrenergic system conferring core posttraumatic stress disorder symptoms such as hyperarousal and reexperiencing. Monoamine oxidase A is one of the key enzymes mediating the turnover of noradrenaline. Here, DNA methylation of the monoamine oxidase A gene exonI/intronI region was investigated for the first time regarding its role in posttraumatic stress disorder risk and severity. Monoamine oxidase A methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells in a total sample of N=652 (441 male) patients with current posttraumatic stress disorder, patients with remitted posttraumatic stress disorder, and healthy probands (comparison group) recruited at 5 centers in Bosnia-Herzegovina, Croatia, and the Republic of Kosovo. Posttraumatic stress disorder severity was measured by means of the Clinician-Administered Posttraumatic Stress Disorder Scale and its respective subscores representing distinct symptom clusters. In the male, but not the female sample, patients with current posttraumatic stress disorder displayed hypermethylation of 3 CpGs (CpG3=43656362; CpG12=43656514; CpG13=43656553, GRCh38.p2 Assembly) as compared with remitted Posttraumatic Stress Disorder patients and healthy probands. Symptom severity (Clinician-Administered Posttraumatic Stress Disorder Scale scores) in male patients with current posttraumatic stress disorder significantly correlated with monoamine oxidase A methylation. This applied particularly to symptom clusters related to reexperiencing of trauma (cluster B) and hyperarousal (cluster D). The present findings suggest monoamine oxidase A gene hypermethylation, potentially resulting in enhanced noradrenergic signalling, as a disease status and severity marker of current posttraumatic stress disorder in males. If replicated, monoamine oxidase A hypermethylation might serve as a surrogate marker of a hyperadrenergic subtype of

GPR43 activation enhances psoriasis-like inflammation through epidermal upregulation of IL-6 and dual oxidase 2 signaling in a murine model.

PubMed

Nadeem, Ahmed; Ahmad, Sheikh F; Al-Harbi, Naif O; El-Sherbeeny, Ahmed M; Al-Harbi, Mohammed M; Almukhlafi, Talal S

2017-05-01

The gut is densely inhabited by commensal bacteria, which metabolize dietary fibers/undigested carbohydrates and produce short-chain fatty acids such as acetate. GPR43 is one of the receptors to sense short-chain fatty acids, and expressed in various immune and non-immune cells. Acetate/GPR43 signaling has been shown to affect various inflammatory diseases through Th17 responses and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) generation. However, no study has previously explored the effects of GPR43 activation during psoriasis-like inflammation. Therefore, this study investigated the effect of acetate/phenylacetamide (GPR43 agonists) on imiquimod induced skin inflammation in mice. Mice were administered phenylacetamide/acetate followed by assessment of skin inflammation, NOXs (NOX-2, NOX-4, dual oxidases), and Th17 related signaling. Our study showed induction of epidermal GPR43 after imiquimod treatment, i.e. psoriasis-like inflammation. Acetate administration in psoriatic mice led to further increase in skin inflammation (ear thickness/myeloperoxidase activity) with concurrent increase in Th17 immune responses and epidermal dual oxidase-2 signaling. Further, topical application of GPR43 agonist, phenylacetamide led to enhanced ear thickness with concomitant epidermal IL-6 signaling as well as dual oxidase-2 upregulation which may be responsible for increased psoriasis-like inflammation. Taken together, dual oxidase-2 and IL-6 play important roles in GPR43-mediated skin inflammation. The current study suggests that GPR43 activation in psoriatic patients may lead to aggravation of psoriatic inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative Activity-Based Flavin-Dependent Oxidase Profiling.

PubMed

Krysiak, Joanna; Breinbauer, Rolf

2017-01-01

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand-protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Multidomain flavin-dependent sulfhydryl oxidases.

PubMed

Coppock, Donald L; Thorpe, Colin

2006-01-01

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Effect of contraceptive steroids on monoamine oxidase activity

PubMed Central

Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

1969-01-01

Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva

2008-02-01

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression.more » In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-{alpha}-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.« less

NADPH Oxidase Plays a Role on Ethanol-Induced Hypertension and Reactive Oxygen Species Generation in the Vasculature.

PubMed

Marchi, Katia Colombo; Ceron, Carla Speroni; Muniz, Jaqueline J; De Martinis, Bruno S; Tanus-Santos, José E; Tirapelli, Carlos Renato

2016-09-01

Investigate the role of NADPH oxidase on ethanol-induced hypertension and vascular oxidative stress. Male Wistar rats were treated with ethanol (20% v/v). Apocynin (10 mg/kg/day, i.p.) prevented ethanol-induced hypertension. The increased contractility of endothelium-intact and endothelium-denuded aortic rings from ethanol-treated rats to phenylephrine was prevented by apocynin. Ethanol consumption increased superoxide anion (O2 (-)) generation and lipid peroxidation and apocynin prevented these responses. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was not prevented by apocynin. Treatment with ethanol did not affect aortic levels of hydrogen peroxide (H2O2) or reduced glutathione (GSH). Ethanol did not alter the activities of xanthine oxidase (XO), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Ethanol increased the expression of Nox1, PKCδ, nNOS, SAPK/JNK and SOD2 in the rat aorta and apocynin prevented these responses. No difference on aortic expression of Nox2, Nox4, p47phox, Nox organizer 1 (Noxo1), eNOS and iNOS was detected after treatment with ethanol. Ethanol treatment did not alter the phosphorylation of SAPK/JNK, p38MAPK, c-Src, Rac1 or PKCδ. The major new finding of our study is that the increased vascular generation of reactive oxygen species (ROS) induced by ethanol is related to increased vascular Nox1/NADPH oxidase expression. This mechanism is involved in vascular dysfunction and hypertension induced by ethanol. Additionally, we conclude that ethanol consumption induces the expression of different proteins that regulate vascular contraction and growth and that NADPH oxidase-derived ROS play a role in such response. The key findings of our study are that ethanol-induced hypertension is mediated by NADPH oxidase. Moreover, increased vascular Nox1 expression is related to the generation of reactive oxygen species (ROS) by ethanol. Finally, ROS induced by ethanol increase the

Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

PubMed Central

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

2014-01-01

Introduction 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. Aims 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. Methods 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. Main Outcome Measures Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Results Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Conclusion 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. PMID:24251665

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

PubMed

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 < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse 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.

An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein.

PubMed

McCready, Jessica; Wong, Daniel S; Burlison, Joseph A; Ying, Weiwen; Jay, Daniel G

2014-04-30

Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

PubMed Central

Legge, M; Duff, G B

1981-01-01

Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information. PMID:6785320

Plasma diamine oxidase levels in pregnancy complicated by threatened abortion.

PubMed

Legge, M; Duff, G B

1981-02-01

Plasma diamine oxidase levels were assayed in 66 patients who presented with pregnancy complicated by threatened abortion. Levels within the normal range were associated with continuing pregnancies, whereas levels below the normal range were associated with subsequent abortion. Among those patients in whom gestation was greater than eight weeks, 66.6% of diamine oxidase levels correctly predicted the pregnancy outcome. Assay of the diamine oxidase levels at eight weeks of gestation or less gave little useful information.

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.).

PubMed

Lin, L S; Varner, J E

1991-05-01

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening."

An amperometric biosensor for glucose detection from glucose oxidase immobilized in polyaniline-polyvinylsulfonate-potassium ferricyanide film.

PubMed

Arslan, Fatma; Beskan, Umut

2014-08-01

In this study, a novel amperometric glucose biosensor with immobilization of glucose oxidase on electrochemically polymerized polyaniline-polyvinylsulphonate-potassium ferricyanide (Pani-Pvs-Fc) films has been accomplished via the entrapment technique. Potassium ferricyanide was used as the mediator. Determination of glucose was carried out by the oxidation of potassium ferrocyanide at 0.3 V vs. Ag/AgCl. The effects of pH and temperature were investigated, and the optimum pH value was found to be 7.5. The storage stability and the operational stability of the enzyme electrode were also studied.

Structure-function relationships in the evolutionary framework of spermine oxidase.

PubMed

Cervelli, Manuela; Salvi, Daniele; Polticelli, Fabio; Amendola, Roberto; Mariottini, Paolo

2013-06-01

Spermine oxidase is a FAD-dependent enzyme that specifically oxidizes spermine, and plays a central role in the highly regulated catabolism of polyamines in vertebrates. The spermine oxidase substrate is specifically spermine, a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function, cellular signalling, nitric oxide synthesis and inhibition of immune responses. The oxidative products of spermine oxidase activity are spermidine, H2O2 and the aldehyde 3-aminopropanal that spontaneously turns into acrolein. In this study the reconstruction of the phylogenetic relationships among spermine oxidase proteins from different vertebrate taxa allowed to infer their molecular evolutionary history, and assisted in elucidating the conservation of structural and functional properties of this enzyme family. The amino acid residues, which have been hypothesized or demonstrated to play a pivotal role in the enzymatic activity, and substrate specificity are here analysed to obtain a comprehensive and updated view of the structure-function relationships in the evolution of spermine oxidase.

Three-dimensional organization of three-domain copper oxidases: A review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukhlistova, N. E., E-mail: amm@ns.crys.ras.ru; Zhukova, Yu. N.; Lyashenko, A. V.

2008-01-15

'Blue' copper-containing proteins are multidomain proteins that utilize a unique redox property of copper ions. Among other blue multicopper oxidases, three-domain oxidases belong to the group of proteins that exhibit a wide variety of compositions in amino acid sequences, functions, and occurrences in organisms. This paper presents a review of the data obtained from X-ray diffraction investigations of the three-dimensional structures of three-domain multicopper oxidases, such as the ascorbate oxidase catalyzing oxidation of ascorbate to dehydroascorbate and its three derivatives; the multicopper oxidase CueO (the laccase homologue); the laccases isolated from the basidiomycetes Coprinus cinereus, Trametes versicolor, Coriolus zonatus, Cerrenamore » maxima, and Rigidoporus lignosus and the ascomycete Melanocarpus albomyces; and the bacterial laccases CotA from the endospore coats of Bacillus subtilis. A comparison of the molecular structures of the laccases of different origins demonstrates that, structurally, these objects are highly conservative. This obviously indicates that the catalytic activity of the enzymes under consideration is characterized by similar mechanisms.« less

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

PubMed

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.

Catalase deficiency may complicate urate oxidase (rasburicase) therapy.

PubMed

Góth, László; Bigler, N William

2007-09-01

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.

[Respiratory oxidases: the enzymes which use most of the oxygen which living things breathe].

PubMed

Toledo-Cuevas, E M

1997-01-01

The respiratory oxidases are the last enzymes of the aerobic respiratory chain. They catalize the reduction of molecular oxygen to water, with generation of an electrochemical gradient useful for the energy demanding cellular processes. Most of the oxidases belong to the heme-copper superfamily. They possess a heme-copper center, constituted of a high spin heme and a CuB center, where the reduction of oxygen takes place and probably where the link to proton pumping is located. The superfamily is divided in two classes: the quinol- and the cytochrome c-oxidases. The latter are divided in the aa3 and the cbb3-type cytochrome c oxidases. The main difference between quinol- and the aa3-type cytochrome c-oxidases is the CuA center, which is absent in the quinol oxidases. The cbb3-type cytochrome oxidases have the binuclear center, but lack the CuA center. They also does not have the classical subunits II and III. These differences seem not to affect the oxygen reduction or the proton pumping. Probably the oxidases have evolved from some denitrification enzymes and prior the photosynthetic process. Also is possible that the cbb3-type cytochrome oxidases or others very similar have been the first oxidases to appear.

Identification of the alternative terminal oxidase of higher plant mitochondria

PubMed Central

Elthon, Thomas E.; McIntosh, Lee

1987-01-01

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed. Images PMID:16593898

Molecular and Biochemical Characterization of a Cytokinin Oxidase from Maize1

PubMed Central

Bilyeu, Kristin D.; Cole, Jean L.; Laskey, James G.; Riekhof, Wayne R.; Esparza, Thomas J.; Kramer, Michelle D.; Morris, Roy O.

2001-01-01

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described. PMID:11154345

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420 Oxidase...

The first mammalian aldehyde oxidase crystal structure: insights into substrate specificity.

PubMed

Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T P; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

2012-11-23

Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents. The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics. Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures. The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.

[Oxygen and the superoxide anion. Modulation of NADPH oxidase?].

PubMed

Delbosc, S; Cristol, J P; Descomps, B; Chénard, J; Sirois, P

2001-01-01

Oxidative stress which results from an imbalance between oxidant production and antioxidant defense mechanisms can promote modifications of lipids, proteins and nucleic acids. This review focuses on the different pathways leading to Reactive Oxygen Species (ROS) production in particular on NADPH oxidase activation. This enzyme is localized in numerous cells including phagocytes and vascular cells and composed of membrane and cytosolic sub-units. The activation of the NADPH oxidase is largely involved in inflammation associated diseases such as asthma, Systemic Inflammatory Response Syndrome and aging associated diseases such as atherosclerosis and neurodeneratives diseases. The modulation of NADPH oxidase could be a way to limit or prevent the development of these diseases.

Aiding and abetting roles of NOX oxidases in cellular transformation

PubMed Central

Block, Karen; Gorin, Yves

2013-01-01

NADPH oxidases of the NADPH oxidase (NOX) family are dedicated reactive oxygen species-generating enzymes that broadly and specifically regulate redox-sensitive signalling pathways that are involved in cancer development and progression. They act at specific cellular membranes and microdomains through the activation of oncogenes and the inactivation of tumour suppressor proteins. In this Review, we discuss primary targets and redox-linked signalling systems that are influenced by NOX-derived ROS, and the biological role of NOX oxidases in the aetiology of cancer. PMID:22918415

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan Xinjuan; Dai Yujie; Li Xing

2011-08-01

and fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.« less

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

USGS Publications Warehouse

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.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

PubMed

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.

Immobilization of xanthine oxidase on a polyaniline silicone support.

PubMed

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.

Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

PubMed

Schenkel, E; Dubois, J G; Helson-Cambier, M; Hanocq, M

1996-02-01

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.

Simple, high-yield purification of xanthine oxidase from bovine milk.

PubMed

Ozer, N; Müftüoglu, M; Ataman, D; Ercan, A; Ogüs, I H

1999-05-13

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

PubMed Central

Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

2011-01-01

Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

PubMed

Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

2011-07-01

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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.

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

PubMed Central

de Oliveira, Jessica Silva Santos; 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. PMID:29641644

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

PubMed

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.

AT₁ receptor and NAD(P)H oxidase mediate angiotensin II-stimulated antioxidant enzymes and mitogen-activated protein kinase activity in the rat hypothalamus.

PubMed

Silva, José; Pastorello, Mariella; Arzola, Jorge; Zavala, Lida E; De Jesús, Sara; Varela, Maider; Matos, María Gabriela; del Rosario Garrido, María; Israel, Anita

2010-12-01

Angiotensin II (AngII) regulates blood pressure and water and electrolyte metabolism through the stimulation of NAD(P)H oxidase and production of reactive oxygen species (ROS) such as O₂⁻, which is metabolised by superoxide dismutase, catalase and glutathione peroxidase. We assessed the role of AT₁ and AT₂ receptors, NAD(P)H oxidase and protein kinase C (PKC) in Ang II-induced sodium and water excretion and their capacity to stimulate antioxidant enzymes in the rat hypothalamus, a brain structure known to express a high density of AngII receptors. Male Sprague-Dawley rats were intracerebroventricularly (ICV) injected with AngII and urinary sodium and water excretion was assessed. Urine sodium concentration was determined using flame photometry. After decapitation the hypothalamus was microdissected under stereomicroscopic control. Superoxide dismutase, catalase and glutathione peroxidase activity were determined spectrophotometrically and extracellular signal-regulated kinase (ERK1/2) activation was analysed by Western blot. AngII-ICV resulted in antidiuresis and natriuresis. ICV administration of losartan, PD123319, apocynin and chelerythrine blunted natriuresis. In hypothalamus, AngII increased catalase, superoxide dismutase and glutation peroxidase activity and ERK1/2 phosphorylation. These actions were prevented by losartan, apocynin and chelerythrine, and increased by PD123319. AT₁ and AT₂ receptors, NAD(P)H oxidase and PKC pathway are involved in the regulation of hydromineral metabolism and antioxidant enzyme activity induced by AngII.

An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

PubMed Central

McCready, Jessica; Wong, Daniel S.; Burlison, Joseph A.; Ying, Weiwen; Jay, Daniel G.

2014-01-01

Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion. PMID:24785146

Structure of caa(3) cytochrome c oxidase--a nature-made enzyme-substrate complex.

PubMed

Noor, Mohamed Radzi; Soulimane, Tewfik

2013-05-01

Aerobic respiration, the energetically most favorable metabolic reaction, depends on the action of terminal oxidases that include cytochrome c oxidases. The latter forms a part of the heme-copper oxidase superfamily and consists of three different families (A, B, and C types). The crystal structures of all families have now been determined, allowing a detailed structural comparison from evolutionary and functional perspectives. The A2-type oxidase, exemplified by the Thermus thermophilus caa(3) oxidase, contains the substrate cytochrome c covalently bound to the enzyme complex. In this article, we highlight the various features of caa(3) enzyme and provide a discussion of their importance, including the variations in the proton and electron transfer pathways.

The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis

PubMed Central

Prieto-Bermejo, Rodrigo; Hernández-Hernández, Angel

2017-01-01

Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed. PMID:28505091

Degradation of oxalate in rats implanted with immobilized oxalate oxidase.

PubMed

Raghavan, K G; Tarachand, U

1986-01-20

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.

Co-immobilization of gold nanoparticles with glucose oxidase to improve bioelectrocatalytic glucose oxidation

NASA Astrophysics Data System (ADS)

Aquino Neto, Sidney; Milton, Ross D.; Crepaldi, Laís B.; Hickey, David P.; de Andrade, Adalgisa R.; Minteer, Shelley D.

2015-07-01

Recently, there has been much effort in developing metal nanoparticle catalysts for fuel oxidation, as well as the development of enzymatic bioelectrocatalysts for fuel oxidation. However, there has been little study of the synergy of hybrid electrocatalytic systems. We report the preparation of hybrid bioanodes based on Au nanoparticles supported on multi-walled carbon nanotubes (MWCNTs) co-immobilized with glucose oxidase (GOx). Mediated electron transfer was achieved by two strategies: ferrocene entrapped within polypyrrole and a ferrocene-modified linear poly(ethylenimine) (Fc-LPEI) redox polymer. Electrochemical characterization of the Au nanoparticles supported on MWCNTs indicate that this catalyst exhibits an electrocatalytic response for glucose even in acidic conditions. Using the redox polymer Fc-LPEI as the mediator, voltammetric and amperometric data demonstrated that these bioanodes can efficiently achieve mediated electron transfer and also indicated higher catalytic currents with the hybrid bioelectrode. From the amperometry, the maximum current density (Jmax) achieved with the hybrid bioelectrode was 615 ± 39 μA cm-2, whereas the bioanode employing GOx only achieved a Jmax of 409 ± 26 μA cm-2. Biofuel cell tests are consistent with the electrochemical characterization, thus confirming that the addition of the metallic species into the bioanode structure can improve fuel oxidation and consequently, improve the power generated by the system.

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

PubMed

Miura, S; Oda, T; Funai, T; Ito, M; Okada, Y; Ichiyama, A

1994-07-01

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

PubMed Central

Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R.; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A. M.; Ruggiero, Carmelina

2014-01-01

Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties. PMID:25372607

Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity.

PubMed

Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

2013-08-01

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

PubMed

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.

Xanthine Oxidase Inhibition by Febuxostat Attenuates Experimental Atherosclerosis in Mice

PubMed Central

Nomura, Johji; Busso, Nathalie; Ives, Annette; Matsui, Chieko; Tsujimoto, Syunsuke; Shirakura, Takashi; Tamura, Mizuho; Kobayashi, Tsunefumi; So, Alexander; Yamanaka, Yoshihiro

2014-01-01

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE−/− mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE−/− mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis. PMID:24686534

Megakaryocyte pathology and bone marrow fibrosis: the lysyl oxidase connection

PubMed Central

Matsuura, Shinobu

2012-01-01

Megakaryocytes (MKs), the platelet precursors, are capable of accumulating DNA greater than a diploid content as part of their cell cycle. MKs have been recognized as mediating fibrosis in a subset of hematologic malignancies, including acute megakaryoblastic leukemia and a subset of myeloproliferative neoplasms. The mechanisms responsible for fibrosis remain only partially understood. Past studies highlighted the role of growth factors in such pathologies, and recently, the protein lysyl oxidase (LOX) has been implicated in proliferation of MKs, ploidy and deposition of fibers. LOX was initially characterized as a protein responsible for the intermolecular cross-linking of elastin and collagen, and in recent years it has been identified as regulator of various pathologies, such as cancer and inflammation. Here, we review recent advances in the understanding of the contribution of MKs to the progression of myelofibrosis, highlighting the newly identified role of LOX. PMID:22767499

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress.

PubMed

Natarajan, Sathish Kumar; Muthukrishnan, Ezhumalai; Khalimonchuk, Oleh; Mott, Justin L; Becker, Donald F

2017-07-01

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ 1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

Interactions of NADPH oxidase, renin-angiotensin-aldosterone system and reactive oxygen species in mequindox-mediated aldosterone secretion in Wistar rats.

PubMed

Huang, Xian-Ju; Wang, Xu; Ihsan, Awais; Liu, Qin; Xue, Xi-Juan; Su, Shi-Jia; Yang, Chun-Hui; Zhou, Wen; Yuan, Zong-Hui

2010-10-05

High doses of mequindox (MEQ) are associated with oxidative stress and pathological toxicity in the kidney. In this study, we demonstrated long term effects of MEQ on intra- or extra-adrenal renin-angiotensin-aldosterone system (RAAS) in vivo. RAAS plays a major role in aldosterone secretion. High doses of MEQ in the diet for 180 days in male rats led to inhibition of intra- and extra-adrenal RAAS, concident with down-regulation of Na(+)/K(+)-ATPase (NAKA) and mineralocorticoid receptor (MR), the downstream of aldosterone action. Significant changes of malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) in kidney were also observed in the high doses (110, 275mg/kg) groups. The mRNA levels of most subunits of NADPH oxidase were significantly upregulated at low doses (25-110mg/kg) but the upregulation was diminished at higher doses in both kidney and adrenal gland, indicating a complicated and contradictory effect of MEQ on NADPH. These results highlight the complex interactions of drug metabolism, RAAS, NADPH oxidase and oxidative stress in response to MEQ-induced tissue toxicity and aldosterone secretion. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Role of reactive oxygen intermediates in the interferon-mediated depression of hepatic drug metabolism and protective effect of N-acetylcysteine in mice.

PubMed

Ghezzi, P; Bianchi, M; Gianera, L; Landolfo, S; Salmona, M

1985-08-01

Interferon (IFN) and IFN inducers are known to depress hepatic microsomal cytochrome P-450 levels, and the liver toxicity of IFN was reported to be lethal in newborn mice. We have observed that administration to mice of IFN and IFN inducers caused a marked increase in liver xanthine oxidase activity. Because this enzyme is well known to produce reactive oxygen intermediates and cytochrome P-450 was reported to be sensitive to the oxidative damage, we have tested the hypothesis that a free radical mechanism could mediate the depression of cytochrome P-450 levels by IFN. Administration to mice of the IFN inducer polyinosinic-polycytidylic acid (2 mg/kg i.p.) caused a 29 to 52% decrease in liver cytochrome P-450. Concomitant p.o. administration of the free radical scavenger, N-acetylcysteine (as a 2.5% solution in drinking water), or the xanthine oxidase inhibitor, allopurinol (100 mg/kg), protected against the IFN-mediated depression of P-450 kg), protected against the IFN-mediated depression of P-450 levels. The results suggest that an increased endogenous generation of free radicals, possibly due to the induction of xanthine oxidase, is implicated in the IFN-mediated depression of liver drug metabolism. The relevance of these data also extends to cases in which this side effect is observed in pathological situations (e.g., viral diseases and administration of vaccines) associated with an induction of IFN.

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

PubMed

Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

2013-06-01

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Determination of Monoamine Oxidase A and B Activity in Long-Term Treated Patients With Parkinson Disease.

PubMed

Müller, Thomas; Riederer, Peter; Grünblatt, Edna

Biogenic amines and monoamine oxidase inhibitors influence peripheral monoamine oxidase enzyme activity in chronic levodopa/dopa decarboxylase inhibitor-treated patients with Parkinson disease. Rasagiline is an irreversible inhibitor of monoamine oxidase B. Safinamide blocks this isoenzyme in a reversible fashion. The aim of this study was to determine monoamine oxidase A (plasma) and B (platelets) enzyme activity in long-term levodopa-treated patients without and with additional oral intake of 50- or 100-mg safinamide or 1-mg rasagiline or first-time intake of rasagiline. Monoamine oxidase A enzyme activity did not differ between all groups. Patients on rasagiline or safinamide showed lower monoamine oxidase-B enzyme activity compared with patients without monoamine oxidase B inhibitor intake. No impact of the number of previous oral levodopa intakes was found. Rasagiline and safinamide did not essentially differ in terms of inhibition of monoamine oxidase B despite their different pharmacology regarding reversibility of monoamine oxidase B inhibition. In view of the observed, considerable heterogeneity of enzyme activities, we suggest to determine activities of monoamine oxidase A and B to reduce the risk for tyramine-induced hypertension and the serotonergic syndrome during chronic therapy with rasagiline or safinamide.

Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

PubMed

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.

Platinum Nanoparticles: Efficient and Stable Catechol Oxidase Mimetics.

PubMed

Liu, Yi; Wu, Haohao; Chong, Yu; Wamer, Wayne G; Xia, Qingsu; Cai, Lining; Nie, Zhihong; Fu, Peter P; Yin, Jun-Jie

2015-09-09

Although enzyme-like nanomaterials have been extensively investigated over the past decade, most research has focused on the peroxidase-like, catalase-like, or SOD-like activity of these nanomaterials. Identifying nanomaterials having oxidase-like activities has received less attention. In this study, we demonstrate that platinum nanoparticles (Pt NPs) exhibit catechol oxidase-like activity, oxidizing polyphenols into the corresponding o-quinones. Four unique approaches are employed to demonstrate the catechol oxidase-like activity exerted by Pt NPs. First, UV-vis spectroscopy is used to monitor the oxidation of polyphenols catalyzed by Pt NPs. Second, the oxidized products of polyphenols are identified by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution mass spectrometry (HRMS) identification. Third, electron spin resonance (ESR) oximetry techniques are used to confirm the O2 consumption during the oxidation reaction. Fourth, the intermediate products of semiquinone radicals formed during the oxidation of polyphenols are determined by ESR using spin stabilization. These results indicate Pt NPs possess catechol oxidase-like activity. Because polyphenols and related bioactive substances have been explored as potent antioxidants that could be useful for the prevention of cancer and cardiovascular diseases, and Pt NPs have been widely used in the chemical industry and medical science, it is essential to understand the potential effects of Pt NPs for altering or influencing the antioxidant activity of polyphenols.

Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase.

PubMed

Castillo, Jaime; Gáspár, Szilveszter; Sakharov, Ivan; Csöregi, Elisabeth

2003-05-01

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.

PROLINE OXIDASES IN HANSENULA SUBPELLICULOSA

PubMed Central

Ling, Chung-Mei; Hedrick, L. R.

1964-01-01

Ling, Chung-Mei (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Proline oxidases in Hansenula subpelliculosa. J. Bacteriol. 87:1462–1470. 1964—Cells of Hansenula subpelliculosa can use l-proline as a carbon and a nitrogen source after a 6- to 8-hr induction period. However, they cannot use l-glutamate as both nitrogen and carbon sources unless the induction period is of several days' duration. Two l-proline oxidases were demonstrated in the mitochondrial preparation of this yeast. One forms the product Δ′-pyrroline-2-carboxylic acid (P2C), which is in equilibrium with α-keto-δ-amino-valeric acid; the other forms the product Δ′-pyrroline-5-carboxylic acid (P5C), which is in equilibrium with glutamic-γ-semialdehyde. The first-mentioned enzyme is induced when l-proline is the carbon source; the second appears to be constitutive, and is probably associated with the use of l-proline as a nitrogen source. The P2C-forming enzyme is specific for the l isomer of proline, and is inactive against l-hydroxyproline. The enzyme activity is at its peak when the mitochondria are prepared from logarithmically grown cells, and is rapidly reduced after cells reach the stationary phase of growth. Kinetic studies with varying concentrations of substrate indicate a Michaelis-Menten constant of 2.45 × 10−2m. Paper chromatographic studies, chemical tests with H2O2, sensitivity to freezing, and spectral measurements indicate that proline oxidase from H. subpelliculosa mitochondria forms a product from l-proline which is like, if not identical to, P2C formed by the action of sheep kidney d-proline oxidase upon dl-proline. The soluble portion of the cell extract contains NAD+ enzymes which use either P2C (α-keto-δ-amino-valeric acid) or P5C (glutamic-γ-semialdehyde) as substrates. No glutamic dehydrogenase activity could be detected when l-glutamic acid and the nicotinamide adenine dinucleotide (NAD+) cofactor were added to the supernatant solution with the

The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy.

PubMed

Elmer, Lawrence W; Bertoni, John M

2008-11-01

The role of monoamine oxidase type B inhibitors in the treatment of Parkinson's disease has expanded with the new monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets. As primary therapy in early disease monoamine oxidase B inhibitors reduce motor disability and delay the need for levodopa. In more advanced disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce 'off' time and may improve gait and freezing. Rasagiline and selegiline oral disintegrating tablets may reduce the safety risks associated with the amfetamine and methamfetamine metabolites of conventional oral selegiline while retaining or improving therapeutic efficacy. Articles were identified by searches of PubMed and searches on the Internet and reviewed. All articles and other referenced materials were retrieved using the keywords 'Parkinson's disease', 'treatment' and 'monoamine oxidase B inhibitor' and were published between 1960 and 2007, with older references selected for historical significance. Only papers published in English were reviewed. Accumulating data support the use of monoamine oxidase B inhibitors as monotherapy for early and mild Parkinson's disease and as adjunctive therapy for more advanced Parkinson's disease with levodopa-associated motor fluctuations. The recently released monoamine oxidase B inhibitor rasagiline and a new formulation, selegiline oral disintegrating tablets, have potential advantages over conventional oral selegiline.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

PubMed

Camadro, J M; Matringe, M; Thome, F; Brouillet, N; Mornet, R; Labbe, P

1995-05-01

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

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

PubMed

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).

Purification of the Alpha Glycerophosphate Oxidase from African Trypanosomes

DTIC Science & Technology

1987-02-02

oxidase (GPO). This enzyme has not been purified or characterize in detail. Inhibition of this enzyme coupled with inhibition of the anaerobic...more manageable afterwards and remained in the procedure although it only slightly increased the yield. The stability of the solubilized enzyme was...whether the detergent was added during the assay or in the solubilization procedure. However, the successful assay for the enzyme was ubiquinol oxidase

Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species▿ †

PubMed Central

Dick, Gregory J.; Torpey, Justin W.; Beveridge, Terry J.; Tebo, Bradley M.

2008-01-01

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase. PMID:18165363

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

PubMed Central

Johnson, John L.; Anderson, Robert S.; Ordal, Erling J.

1970-01-01

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA–DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA–DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid. PMID:5413826

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate].

PubMed

Medentsev, A G; Arinbasarova, A Iu; Smirnova, N M; Akimenko, V K

2004-01-01

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

Systemic Manifestations in Pyridox(am)ine 5'-Phosphate Oxidase Deficiency.

PubMed

Guerriero, Réjean M; Patel, Archana A; Walsh, Brian; Baumer, Fiona M; Shah, Ankoor S; Peters, Jurriaan M; Rodan, Lance H; Agrawal, Pankaj B; Pearl, Phillip L; Takeoka, Masanori

2017-11-01

Pyridoxine is converted to its biologically active form pyridoxal-5-phosphate (P5P) by the enzyme pyridox(am)ine 5'-phosphate oxidase and serves as a cofactor in nearly 200 reactions in the central nervous system. Pyridox(am)ine 5'-phosphate oxidase deficiency leads to P5P dependent epilepsy, typically a neonatal- or infantile-onset epileptic encephalopathy treatable with P5P or in some cases, pyridoxine. Following identification of retinopathy in a patient with pyridox(am)ine 5'-phosphate oxidase deficiency that was reversible with P5P therapy, we describe the systemic manifestations of pyridox(am)ine 5'-phosphate oxidase deficiency. A series of six patients with homozygous mutations of PNPO, the gene coding pyridox(am)ine 5'-phosphate oxidase, were evaluated in our center over the course of two years for phenotyping of neurological and systemic manifestations. Five of six were born prematurely, three had anemia and failure to thrive, and two had elevated alkaline phosphatase. A movement disorder was observed in two children, and a reversible retinopathy was observed in the most severely affected infant. All patients had neonatal-onset epilepsy and were on a continuum of developmental delay to profound encephalopathy. Electroencephalographic features included background slowing and disorganization, absent sleep features, and multifocal and generalized epileptiform discharges. All the affected probands carried a homozygous PNPO mutation (c.674 G>T, c.686 G>A and c.352G>A). In addition to the well-described epileptic encephalopathy, pyridox(am)ine 5'-phosphate oxidase deficiency causes a range of neurological and systemic manifestations. A movement disorder, developmental delay, and encephalopathy, as well as retinopathy, anemia, and failure to thrive add to the broadening clinical spectrum of P5P dependent epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase.

PubMed

Kuchar, Manuela; Neuber, Christin; Belter, Birgit; Bergmann, Ralf; Lenk, Jens; Wodtke, Robert; Kniess, Torsten; Steinbach, Jörg; Pietzsch, Jens; Löser, Reik

2018-01-01

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N -succinimidyl 4-[ 18 F]fluorobenzoate ([ 18 F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo , their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18 F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase

PubMed Central

Kuchar, Manuela; Neuber, Christin; Belter, Birgit; Bergmann, Ralf; Lenk, Jens; Wodtke, Robert; Kniess, Torsten; Steinbach, Jörg; Pietzsch, Jens; Löser, Reik

2018-01-01

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

Protein structural development of threadfin bream ( Nemipterus spp.) surimi gels induced by glucose oxidase.

PubMed

Wang, Lei; Fan, Daming; Fu, Lulu; Jiao, Xidong; Huang, Jianlian; Zhao, Jianxin; Yan, Bowen; Zhou, Wenguo; Zhang, Wenhai; Ye, Weijian; Zhang, Hao

2018-01-01

This study investigated the effect of glucose oxidase on the gel properties of threadfin bream surimi. The gel strength of surimi increased with the addition of 0.5‰ glucose oxidase after two-step heating. Based on the results of the chemical interactions, the hydrophobic interaction and disulfide bond of glucose oxidase-treated surimi samples increased compared with the control samples at the gelation temperature and gel modori temperature. The surface hydrophobicity of samples with glucose oxidase and glucose increased significantly ( p < 0.05) and total sulfhydryl groups decreased significantly ( p < 0.05). The analysis of Raman spectroscopy shows that the addition of glucose oxidase induced more α-helixes to turn into a more elongated random and flocculent structure. Glucose oxidase changes the secondary structure of the surimi protein, making more proteins depolarize and stretch and causing actomyosin to accumulate to each other, resulting in the formation of surimi gel.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

PubMed Central

Thomazella, Daniela P T; Teixeira, Paulo José P L; Oliveira, Halley C; Saviani, Elzira E; Rincones, Johana; Toni, Isabella M; Reis, Osvaldo; Garcia, Odalys; Meinhardt, Lyndel W; Salgado, Ione; Pereira, Gonçalo A G

2012-01-01

The tropical pathogen Moniliophthora perniciosa causes witches’ broom disease in cacao. As a hemibiotrophic fungus, it initially colonizes the living host tissues (biotrophic phase), and later grows over the dead plant (necrotrophic phase). Little is known about the mechanisms that promote these distinct fungal phases or mediate the transition between them. An alternative oxidase gene (Mp-aox) was identified in the M. perniciosa genome and its expression was analyzed througout the fungal life cycle. In addition, the effects of inhibitors of the cytochrome-dependent respiratory chain (CRC) and alternative oxidase (AOX) were evaluated on the in vitro development of M. perniciosa. Larger numbers of Mp-aox transcripts were observed in the biotrophic hyphae, which accordingly showed elevated sensitivity to AOX inhibitors. More importantly, the inhibition of CRC prevented the transition from the biotrophic to the necrotrophic phase, and the combined use of a CRC and AOX inhibitor completely halted fungal growth. On the basis of these results, a novel mechanism is presented in which AOX plays a role in the biotrophic development of M. perniciosa and regulates the transition to its necrotrophic stage. Strikingly, this model correlates well with the infection strategy of animal pathogens, particularly Trypanosoma brucei, which uses AOX as a strategy for pathogenicity. PMID:22443281

NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

PubMed Central

Peck, Ammon B.; Khan, Saeed R.

2013-01-01

A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

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

PubMed

Butorina, N N

2010-01-01

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

Heterologous production and characterization of two glyoxal oxidases from Pycnoporus cinnabarinus

Treesearch

Marianne Daou; François Piumi; Daniel Cullen; Eric Record; Craig B. Faulds

2016-01-01

The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium...

Spermidine-mediated hydrogen peroxide signaling enhances the antioxidant capacity of salt-stressed cucumber roots.

PubMed

Wu, Jianqiang; Shu, Sheng; Li, Chengcheng; Sun, Jin; Guo, Shirong

2018-07-01

Hydrogen peroxide (H 2 O 2 ) is a key signaling molecule that mediates a variety of physiological processes and defense responses against abiotic stress in higher plants. In this study, our aims are to clarify the role of H 2 O 2 accumulation induced by the exogenous application of spermidine (Spd) to cucumber (Cucumis sativus) seedlings in regulating the antioxidant capacity of roots under salt stress. The results showed that Spd caused a significant increase in endogenous polyamines and H 2 O 2 levels, and peaked at 2 h after salt stress. Spd-induced H 2 O 2 accumulation was blocked under salt stress by pretreatment with a H 2 O 2 scavenger and respective inhibitors of cell wall peroxidase (CWPOD; EC: 1.11.1.7), polyamine oxidase (PAO; EC: 1.5.3.11) and NADPH oxidase (NOX; EC: 1.6.3.1); among these three inhibitors, the largest decrease was found in response to the addition of the inhibitor of polyamine oxidase. In addition, we observed that exogenous Spd could increase the activities of the enzymes superoxide dismutase (SOD; EC: 1.15.1.1), peroxidase (POD; EC: 1.11.1.7) and catalase (CAT; EC: 1.11.1.6) as well as the expression of their genes in salt-stressed roots, and the effects were inhibited by H 2 O 2 scavengers and polyamine oxidase inhibitors. These results suggested that, by regulating endogenous PAs-mediated H 2 O 2 signaling in roots, Spd could enhance antioxidant enzyme activities and reduce oxidative damage; the main source of H 2 O 2 was polyamine oxidation, which was associated with improved tolerance and root growth recovery of cucumber under salt stress. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

PubMed Central

Li, Q.; Ritzel, R. G.; McLean, LLT.; McIntosh, L.; Ko, T.; Bertrand, H.; Nargang, F. E.

1996-01-01

Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1. PMID:8770590

Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments

PubMed Central

Liu, Wei; Glunde, Kristine; Bhujwalla, Zaver M.; Raman, Venu; Sharma, Anit; Phang, James M.

2012-01-01

Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation co-localized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low-glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase (AMPK) activation, but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics. PMID:22609800

Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gamache, D.A.; Kornberg, L.J.; Bartolf, M.

1986-05-01

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-/sup 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/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimalmore » concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 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/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.« less

Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

PubMed

Wu, K; Li, L; Gage, D A; Zeevaart, J A

1996-02-01

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.

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

PubMed

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.

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

PubMed Central

Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

2014-01-01

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed. PMID:24466040

Phenol oxidase activity in secondary transformed peat-moorsh soils

NASA Astrophysics Data System (ADS)

Styła, K.; Szajdak, L.

2009-04-01

The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites of investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at λmax=525 nm with catechol as substrate by method of Perucci

Mitochondrial terminal alternative oxidase and its enhancement by thermal stress in the coral symbiont Symbiodinium

NASA Astrophysics Data System (ADS)

Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.

2014-06-01

A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means of reducing oxidative stress in mitochondria, as well as a potential mechanism for ameliorating thermal and other physiological stressors. Here, the presence of an active AOX in cultured Symbiodinium was assayed by the response of oxygen consumption to the AOX inhibitor salicylhydroxamic acid (SHAM) and the COX inhibitor cyanide (CN). CN-insensitive, SHAM-sensitive oxygen consumption was found to account for a large portion (26 %) of Symbiodinium dark respiration and is consistent with high levels of AOX activity. This experimental evidence of the existence of a previously unreported terminal oxidase was further corroborated by analysis of publicly available Symbiodinium transcriptome data. The potential for enhanced AOX expression to play a compensatory role in mediating thermal stress was supported by inhibitor assays of cultured Symbiodinium at low (18 °C), moderate (26 °C), and high (32 °C) temperature conditions. Maximum capacity of the putative AOX pathway as a proportion of total dark oxygen consumption was found to increase from 26 % at 26 °C to 45 % and 53 % at 18 °C and 32 °C, respectively, when cells were acclimated to the treatment temperatures. Cells assayed at 18 and 32 °C without acclimation exhibited either the same or lower AOX capacity as controls, suggesting that the AOX protein is upregulated under temperature stress. The physiological implications for the presence of AOX in the coral/algal symbiosis and its potential role in response to many forms of biotic and abiotic stress, particularly oxidative stress, are discussed.

Design, synthesis and molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors.

PubMed

Shi, Da-Hua; Huang, Wei; Li, Chao; Liu, Yu-Wei; Wang, Shi-Fan

2014-03-21

A series of aloe-emodin derivatives were synthesized and evaluated as xanthine oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant inhibitory activities against xanthine oxidase. The compound 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the best xanthine oxidase inhibitory activity with IC50 of 2.79 μM. Lineweaver-Burk plot analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The docking study revealed that the molecule A1 had strong interactions with the active site of xanthine oxidase and this result was in agreement with kinetic study. Consequently, compound A1 is a new-type candidate for further development for the treatment of gout. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Supplementary biochemical tests useful for the differentiation of oxidase positive staphylococci.

PubMed

Stepanović, Srdjan; Dakić, Ivana; Hauschild, Tomasz; Vuković, Dragana; Morrison, Donald; Jezek, Petr; Cirković, Ivana; Petrás, Petr

2007-06-01

Differentiation of the oxidase positive staphylococci, Staphylococcus sciuri, Staphylococcus lentus, Staphylococcus vitulinus and Staphylococcus fleurettii, based on tributyrin, urease, caseinase, gelatinase and DNase activity is described. These tests may be used for preliminary identification of oxidase positive isolates of staphylococci resulting in more accurate identification of these species.

NADPH oxidases: new kids on the block.

PubMed

Geiszt, Miklós

2006-07-15

Reactive oxygen species (ROS) play a pivotal role in many physiological processes including host defense, hormone biosynthesis, fertilization and cellular signaling. Altered production of ROS has been implicated in the development of immunodeficiency, hypothyroidism and cardiovascular pathologies. In the last few years, several enzymes were identified at the molecular level, which are now thought to be responsible for ROS production observed in diverse tissues. These enzymes show a high degree of homology to the phagocytic NADPH oxidase and are now designated the Nox family of NADPH oxidases. This review updates our knowledge on six new members of the Nox family: Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2.

Proline Oxidase (POX) as A Target for Cancer Therapy.

PubMed

Kononczuk, Joanna; Czyzewska, Urszula; Moczydlowska, Joanna; Surażyński, Arkadiusz; Palka, Jerzy; Miltyk, Wojciech

2015-01-01

Proline dehydrogenase/proline oxidase (PRODH/POX) is an enzyme catalyzing the first step of proline degradation, during which ROS and/or ATP is generated. POX is widely distributed in living organisms and is responsible for a number of regulatory processes such as redox homeostasis, osmotic adaptation, cell signaling and oxidative stress. Recent data provided evidence that POX plays an important role in carcinogenesis and tumor growth. POX may induce apoptosis in both intrinsic and extrinsic way. Due to ROS generation, POX may induce caspase-9 activity, which mediates mitochondrial apoptosis (intrinsic apoptosis pathway). POX can also stimulate TRAIL (tumor necrosis factorrelated apoptosis inducing ligand) and DR5 (death receptor 5) expression, resulting in cleavage of procaspase-8 and thus extrinsic apoptotic pathway. However, this tumor suppressor in certain environmental conditions may act as a prosurvival factor. Genotoxic, inflammatory and metabolic stress may switch POX from tumor growth inhibiting to tumor growth supporting factor. The potential mechanisms which may regulate switching of POX mode are discussed in this review.

A dityrosine network mediated by dual oxidase and peroxidase influences the persistence of Lyme disease pathogens within the vector.

PubMed

Yang, Xiuli; Smith, Alexis A; Williams, Mark S; Pal, Utpal

2014-05-02

Ixodes scapularis ticks transmit a wide array of human and animal pathogens including Borrelia burgdorferi; however, how tick immune components influence the persistence of invading pathogens remains unknown. As originally demonstrated in Caenorhabditis elegans and later in Anopheles gambiae, we show here that an acellular gut barrier, resulting from the tyrosine cross-linking of the extracellular matrix, also exists in I. scapularis ticks. This dityrosine network (DTN) is dependent upon a dual oxidase (Duox), which is a member of the NADPH oxidase family. The Ixodes genome encodes for a single Duox and at least 16 potential peroxidase proteins, one of which, annotated as ISCW017368, together with Duox has been found to be indispensible for DTN formation. This barrier influences pathogen survival in the gut, as an impaired DTN in Doux knockdown or in specific peroxidase knockdown ticks, results in reduced levels of B. burgdorferi persistence within ticks. Absence of a complete DTN formation in knockdown ticks leads to the activation of specific tick innate immune pathway genes that potentially resulted in the reduction of spirochete levels. Together, these results highlighted the evolution of the DTN in a diverse set of arthropod vectors, including ticks, and its role in protecting invading pathogens like B. burgdorferi. Further understanding of the molecular basis of tick innate immune responses, vector-pathogen interaction, and their contributions in microbial persistence may help the development of new targets for disrupting the pathogen life cycle.

Effect of mitoguazone on polyamine oxidase activity in rat liver.

PubMed

Ferioli, Maria Elena; Berselli, Debora; Caimi, Samuela

2004-12-01

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

A Mycobacterium tuberculosis Cytochrome bd Oxidase Mutant Is Hypersensitive to Bedaquiline

PubMed Central

Hartman, Travis E.

2014-01-01

ABSTRACT The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. PMID:25028424

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

PubMed

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.

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Direct Measurement of Cyclic Current-Voltage Responses of Integral Membrane Proteins at a Self-Assembled Lipid-Bilayer-Modified Electrode: Cytochrome f and Cytochrome c Oxidase

NASA Astrophysics Data System (ADS)

Salamon, Z.; Hazzard, J. T.; Tollin, G.

1993-07-01

Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

Safety assessment of bacterial choline oxidase protein introduced in transgenic crops for tolerance against abiotic stress.

PubMed

Singh, Abinav K; Singh, Bhanu P; Prasad, G B K S; Gaur, Shailendra N; Arora, Naveen

2008-12-24

Genetically modified crops have resistance to abiotic stress by introduction of choline oxidase protein. In the present study, the safety of choline oxidase protein derived from Arthrobacter globiformis was assessed for toxicity and allergenicity. The protein was stable at 90 degrees C for 1 h. Toxicity studies of choline oxidase in mice showed no significant difference (p > 0.05) from control in terms of growth, body weight, food consumption, and blood biochemical indices. Histology of gut tissue of mice fed protein showed normal gastric mucosal lining and villi in jejunum and ileum sections. Specific IgE in serum and IL-4 release in splenic culture supernatant were low in choline oxidase treated mice, comparable to control. Intravenous challenge with choline oxidase did not induce any adverse reaction, unlike ovalbumin group mice. Histology of lung tissues from choline oxidase sensitized mice showed normal airways, whereas ovalbumin-sensitized mice showed inflamed airways with eosinophilic infiltration and bronchoconstriction. ELISA carried out with food allergic patients' sera revealed no significant IgE affinity with choline oxidase. Also, choline oxidase did not show any symptoms of toxicity and allergenicity in mice.

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

PubMed Central

Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

2000-01-01

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

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

PubMed

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.

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

PubMed

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.

An NADPH Oxidase RBOH Functions in Rice Roots during Lysigenous Aerenchyma Formation under Oxygen-Deficient Conditions

PubMed Central

Yoshioka, Miki; Fukazawa, Aya; Nishizawa, Naoko K.

2017-01-01

Reactive oxygen species (ROS) produced by the NADPH oxidase, respiratory burst oxidase homolog (RBOH), trigger signal transduction in diverse biological processes in plants. However, the functions of RBOH homologs in rice (Oryza sativa) and other gramineous plants are poorly understood. Ethylene induces the formation of lysigenous aerenchyma, which consists of internal gas spaces created by programmed cell death of cortical cells, in roots of gramineous plants under oxygen-deficient conditions. Here, we report that, in rice, one RBOH isoform (RBOHH) has a role in ethylene-induced aerenchyma formation in roots. Induction of RBOHH expression under oxygen-deficient conditions was greater in cortical cells than in cells of other root tissues. In addition, genes encoding group I calcium-dependent protein kinases (CDPK5 and CDPK13) were strongly expressed in root cortical cells. Coexpression of RBOHH with CDPK5 or CDPK13 induced ROS production in Nicotiana benthamiana leaves. Inhibitors of RBOH activity or cytosolic calcium influx suppressed ethylene-induced aerenchyma formation. Moreover, knockout of RBOHH by CRISPR/Cas9 reduced ROS accumulation and inducible aerenchyma formation in rice roots. These results suggest that RBOHH-mediated ROS production, which is stimulated by CDPK5 and/or CDPK13, is essential for ethylene-induced aerenchyma formation in rice roots under oxygen-deficient conditions. PMID:28351990

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.

PubMed

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

Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

USDA-ARS?s Scientific Manuscript database

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both Myrothecium verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of...

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing.

PubMed

Zhou, Xuechou; Tan, Bingcan; Zheng, Xinyu; Kong, Dexian; Li, Qinglu

2015-11-15

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5-5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose. Copyright © 2015 Elsevier Inc. All rights reserved.

Towards rational therapy with monoamine oxidase inhibitors.

PubMed

Tyrer, P

1976-04-01

A rational approach to the use of monoamine oxidase inhibitors (MAOIs) is outlined. Patients suitable for treatment cannot be classified adequately using conventional diagnostic labels. They include those with primary symptoms of hypochondriasis, agoraphobia and social phobias, irritability, somatic anxiety and anergia; those with primary depressed mood, guilt, ideas of reference and personality disorders seldom respond. There is great variation in the interval between the first administration of these drugs and clinical response, and this may account for the inconsistencies in published trials. The type of drug and its dose may affect rate of response, as may biochemical factors, including acetylator and monoamine oxidase status. To obtain maximum benefit, a course of therapy with MAOIs should last for several months.

Mammalian monoamine-oxidizing enzymes, with special reference to benzylamine oxidase in human tissues.

PubMed

Lewinsohn, R

1984-01-01

A review is presented of the monoamine-oxidizing enzymes with special reference to the activity of benzylamine oxidase (BzAO) in human tissues. Methods of study of amine oxidases, properties (chiefly of BzAO) and some problems concerning substrate and inhibitor specificity and multiple forms of monoamine oxidase (MAO) are surveyed. The substrate specificity of human plasma BzAO is compared with that of amine-oxidizing enzymes in plasma or serum of other species. Correlations of plasma BzAO and platelet MAO activity with clinical findings are discussed. The distribution of amine oxidase activities in solid human tissues is reviewed, in particular BzAO in blood vessels and richly-vascularized tissues, as well as kinetic constants and altered patterns of activity of BzAO in human atherosclerosis. Activities of the amine oxidases in non-vascular smooth muscle, in cultured cells, and in various tissues related to human gestation, are discussed. The present knowledge of BzAO is discussed in terms of its possible clinical relevance to several human disease states, and the importance of the enzyme in the human body.

Herbivore-plant interactions: mixed-function oxidases and secondary plant substances.

PubMed

Brattsten, L B; Wilkinson, C F; Eisner, T

1977-06-17

The mixed-function oxidases of a polyphagous insect larva (the southern armyworm, Spodoptera eridania) were found to be induced by a diversity of secondary plant substances. The induction proceeds rapidly and in response to a small quantity of secondary substance. Following induction, the larva is less susceptible to dietary poisoning. It is argued that mixed-function oxidases play a major role in protecting herbivores against chemical stress from secondary plant substances.

Snake Venom L-Amino Acid Oxidases: Trends in Pharmacology and Biochemistry

PubMed Central

Izidoro, Luiz Fernando M.; Sobrinho, Juliana C.; Mendes, Mirian M.; Costa, Tássia R.; Grabner, Amy N.; Rodrigues, Veridiana M.; da Silva, Saulo L.; Zanchi, Fernando B.; Zuliani, Juliana P.; Fernandes, Carla F. C.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.

2014-01-01

L-amino acid oxidases are enzymes found in several organisms, including venoms of snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. L-amino acid oxidases exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage, and cytotoxicity. These proteins present a high biotechnological potential for the development of antimicrobial, antitumor, and antiprotozoan agents. This review provides an overview of the biochemical properties and pharmacological effects of snake venom L-amino acid oxidases, their structure/activity relationship, and supposed mechanisms of action described so far. PMID:24738050

A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

PubMed Central

Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina

2017-01-01

ABSTRACT We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.−) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of

Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases.

PubMed

Rahman, Taufiq; Rahmatullah, Mohammed

2010-01-15

Several studies have revealed piperine and a few related compounds as potent inhibitors of monoamine oxidases without delineating the underlying mechanism. Using in silico modelling, we propose a structural basis of such activity by showing that these compounds can successfully dock into the inhibitor binding pockets of human monoamine oxidase isoforms with predicted affinities comparable to some known inhibitors. The results therefore suggest that piperine can be a promising lead for developing novel monoamine oxidase inhibitors. Copyright 2009 Elsevier Ltd. All rights reserved.

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

PubMed

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 (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) 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

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

PubMed Central

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

2012-01-01

Introduction 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. Aims 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. Methods 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 p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion 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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun

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 thatmore » 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.« less

Decolorization and biodegradation of remazol brilliant blue R by bilirubin oxidase.

PubMed

Liu, Youxun; Huang, Juan; Zhang, Xiaoyu

2009-12-01

The dye-decolorizing potential of bilirubin oxidase (BOX) was demonstrated for an anthraquinone dye, remazol brilliant blue R (RBBR). The dye was decolorized 40% within 4 h by the BOX alone, whereas it was more efficient in the presence of 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), showing 91.5% decolorization within 25 min. The effects of operational parameters on decolorization were examined. The results showed that the decolorization efficiency decreased with increasing RBBR concentration, and a marked inhibition effect was exhibited when the dye concentrations were above 100 mg l(-1). The optimum temperature for enzymatic decolorization was 40 degrees C. BOX showed efficient decolorization of the dye with a wide pH range of 5-8.5. The maximum decolorization activity occurred at pH 8 with ABTS and at pH 5 without ABTS. Analysis of RBBR ultraviolet and visible (UV-VIS) spectra after BOX treatment indicated that the decolorization of RBBR was due to biodegradation. Our results suggested that ABTS can serve as an electron mediator to facilitate the oxidation of RBBR, and the BOX-ABTS mediator-involved dye decolorization mechanism was similar to that of laccase. Operation over a wide range of pH and efficient decolorization suggested that the BOX can be used to decolorize synthetic dyes from effluents, especially for anthraquinonic dyes.

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

PubMed Central

Lee, Mark J.; Liu, Hong; Barker, Bridget M.; Snarr, Brendan D.; Gravelat, Fabrice N.; Al Abdallah, Qusai; Gavino, Christina; Baistrocchi, Shane R.; Ostapska, Hanna; Xiao, Tianli; Ralph, Benjamin; Solis, Norma V.; Lehoux, Mélanie; Baptista, Stefanie D.; Thammahong, Arsa; Cerone, Robert P.; Kaminskyj, Susan G. W.; Guiot, Marie-Christine; Latgé, Jean-Paul; Fontaine, Thierry; Vinh, Donald C.; Filler, Scott G.; Sheppard, Donald C.

2015-01-01

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs. PMID:26492565

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

PubMed

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.

Function of the Pyruvate Oxidase-Lactate Oxidase Cascade in Interspecies Competition between Streptococcus oligofermentans and Streptococcus mutans

PubMed Central

Liu, Lei

2012-01-01

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H2O2 production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H2O2 in the earlier growth phase and log phase, while Lox mainly contributed to H2O2 production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H2O2 can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H2O2 production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H2O2 formation so as to win the interspecies competition. PMID:22287002

The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

PubMed

Wongnate, Thanyaporn; Chaiyen, Pimchai

2013-07-01

Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. © 2013 The Authors Journal compilation © 2013 FEBS.

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

PubMed

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.

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

PubMed

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.

Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode.

PubMed

Khayyami, M; Johansson, G; Kriz, D; Xie, B; Larsson, P O; Danielsson, B

1996-06-01

An electron transfer mediator, 8-dimethylamino-2,3-benzophenoxazine (Meldola Blue), dissolved in the carrier solution in a flow-injection system, was found to reduce the oxidation potential for hydrogen peroxide from 600-1200 mV without mediator to-100 mV vs. Ag/AgCl with the mediator present. The very low background current of reticulated vitreous carbon (RVC) at this potential makes it possible to detect very low levels of hydrogen peroxide or glucose. Glucose oxidase was covalently coupled with carbodiimide to RVC, and the RVC was formed into a column inserted in a flow-injection system. The calibration curve was linear from 30 nM to 10 microM glucose with 5 microM mediator. At higher mediator concentrations, the linear range was extended to 1000 microM, but with a much higher background current. The sample throughput was about 60 h(-1). The current response decreased to 50% of the original response after 20 days. The coulometric yield was high because the sample was pumped through the pores of the RVC. It was 16% and 55% at a flow rate of 1 ml min(-1) at mediator concentrations of 5 and 50 microM respectively.

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

PubMed

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.

Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22{sup phox} expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chaoyun; He, Yanhao; Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Disease, Ministry of Education, Xi'an, Shaanxi 710061

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 ofmore » 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{sup 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{sup 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.« less

Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase

PubMed Central

Jiang, F; Zhang, G; Hashimoto, I; Kumar, B S; Bortolotto, S; Morrison, W A; Dusting, G J

2008-01-01

Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth. PMID:19012731

Recent advances in Parkinson's disease therapy: use of monoamine oxidase inhibitors.

PubMed

Henchcliffe, Claire; Schumacher, H Christian; Burgut, F Tuna

2005-11-01

Monoamine oxidase inhibitors inhibit dopamine metabolism and are therefore effective in treating Parkinson's disease, a condition associated with progressive striatal dopamine deficiency secondary to degeneration of dopaminergic neurons in the substantia nigra. Selegiline is currently the most widely used monoamine oxidase-B inhibitor for Parkinson's disease, but has a low and variable bioavailability, and is metabolized to L-methamphetamine and L-amphetamine that carry a risk for potential neurotoxicity. There are two new approaches that circumvent these potential disadvantages. First, selegiline orally disintegrating tablets provide a novel delivery form of selegiline, avoiding first pass metabolism by rapid absorption through the oral mucosa, thus leading to significantly lower plasma concentrations of L-metamphetamine and L-amphetamine. Selegiline orally disintegrating tablets prove to be clinically effective and safe in patients with moderately advanced Parkinson's disease. Second, rasagiline is a new monoamine oxidase inhibitor, without known neurotoxic metabolites. In large clinical trials, rasagiline proves effective as monotherapy in early Parkinson's disease, as well as adjunctive therapy to levodopa in advanced disease. Clinical data suggest, in addition, a disease-modifying effect of rasagiline that may correlate with neuroprotective activity of monoamine oxidase-B inhibitors in animal models of Parkinson's disease.

Insights into proton translocation in cbb3 oxidase from MD simulations.

PubMed

Carvalheda, Catarina A; Pisliakov, Andrei V

2017-05-01

Heme-copper oxidases are membrane protein complexes that catalyse the final step of the aerobic respiration, namely the reduction of oxygen to water. The energy released during catalysis is coupled to the active translocation of protons across the membrane, which contributes to the establishment of an electrochemical gradient that is used for ATP synthesis. The distinctive C-type (or cbb 3 ) cytochrome c oxidases, which are mostly present in proteobacteria, exhibit a number of unique structural and functional features, including high catalytic activity at low oxygen concentrations. At the moment, the functioning mechanism of C-type oxidases, in particular the proton transfer/pumping mechanism presumably via a single proton channel, is still poorly understood. In this work we used all-atom molecular dynamics simulations and continuum electrostatics calculations to obtain atomic-level insights into the hydration and dynamics of a cbb 3 oxidase. We provide the details of the water dynamics and proton transfer pathways for both the "chemical" and "pumped" protons, and show that formation of protonic connections is strongly affected by the protonation state of key residues, namely H243, E323 and H337. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

PubMed Central

Frisse, Andrea; Pimenta, Maria João; Lange, Theo

2003-01-01

Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

USDA-ARS?s Scientific Manuscript database

Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ pump

PubMed Central

Tazzeo, T; Worek, F; Janssen, LJ

2009-01-01

Background and purpose: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. Experimental approach: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca2+ pump activity was assessed by Ca2+ uptake into smooth muscle microsomes. Key results: Contractions to acetylcholine were markedly enhanced by DPI (10−4 M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC50∼8 × 10−6 M and 6 × 10−7 M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca2+ pump. Direct measurement of microsomal Ca2+ uptake revealed that DPI modestly inhibits the internal Ca2+ pump. Conclusions and implications: DPI inhibits cholinesterase activity and the internal Ca2+ pump in tracheal smooth muscle. PMID:19788497

Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.

PubMed

Kim, Mi Jin; Nagy, Laura E; Park, Pil-Hoon

2014-09-01

Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-κB pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Increased xanthine oxidase during labour--implications for oxidative stress.

PubMed

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.

A Conserved Steroid Binding Site in Cytochrome c Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Ling; Mills, Denise A.; Buhrow, Leann

2010-09-02

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

Fluorescence ELISA based on glucose oxidase-mediated fluorescence quenching of quantum dots for highly sensitive detection of Hepatitis B.

PubMed

Wu, Yunqing; Zeng, Lifeng; Xiong, Ying; Leng, Yuankui; Wang, Hui; Xiong, Yonghua

2018-05-01

Herein, we present a novel sandwich fluorescence enzyme linked immunosorbent assay (ELISA) for highly sensitive detection of Hepatitis B virus surface antigen (HBsAg) based on glucose oxidase (GOx)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (MPA-QDs). In this system, hydrogen peroxide (H 2 O 2 ) sensitive MPA-QDs was used as a signal output, and glucose oxidase (GOx) was used as label which can generate H 2 O 2 via catalytic oxidation of glucose. The proposed method showed dynamic linear detection of HBsAg both in the range of 47pgmL -1 ~ 380pgmL -1 and 0.75ngmL -1 ~ 12.12ngmL -1 . The detection limit of the proposed fluorescence ELISA was 1.16pgmL -1 , which was approximately 430-fold lower than that of horseradish peroxidase (HRP)-based conventional ELISA. The average recoveries for HBsAg-spiked serum samples ranged from 98.0% to 126.8% with the relative standard derivation below 10%, thus indicating acceptable precision and high reproducibility of the proposed fluorescence ELISA for HBsAg detection. Additionally, the developed method showed no false positive results analyzing 35 real HBsAg-negative serum samples, and exhibited excellent agreement (R 2 =0.9907) with a commercial time-resolved fluorescence immunoassay (TRFIA) kit for detecting 31 HBsAg-positive serum samples. In summary, the proposed method based on fluorescence quenching of H 2 O 2 sensitive QDs is considerably to be an excellent biodetection platform with ultrahigh sensitivity, good accuracy and excellent reliability. Copyright © 2018 Elsevier B.V. All rights reserved.

Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

PubMed

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)).

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro.

PubMed

Choi, Young Ji; Kim, Da Hye; Kim, Sang Jun; Kim, Ju; Jeong, Seung-Il; Chung, Chang Ho; Yu, Kang-Yeol; Kim, Seon-Young

2014-07-17

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation. Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl4) with or without decursin for 4weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs. Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl4 treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3-Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver. Decursin blocked CCl4-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Monoamine oxidase inactivation: from pathophysiology to therapeutics.

PubMed

Bortolato, Marco; Chen, Kevin; Shih, Jean C

2008-01-01

Monoamine oxidases (MAOs) A and B are mitochondrial bound isoenzymes which catalyze the oxidative deamination of dietary amines and monoamine neurotransmitters, such as serotonin, norepinephrine, dopamine, beta-phenylethylamine and other trace amines. The rapid degradation of these molecules ensures the proper functioning of synaptic neurotransmission and is critically important for the regulation of emotional behaviors and other brain functions. The byproducts of MAO-mediated reactions include several chemical species with neurotoxic potential, such as hydrogen peroxide, ammonia and aldehydes. As a consequence, it is widely speculated that prolonged excessive activity of these enzymes may be conducive to mitochondrial damages and neurodegenerative disturbances. In keeping with these premises, the development of MAO inhibitors has led to important breakthroughs in the therapy of several neuropsychiatric disorders, ranging from mood disorders to Parkinson's disease. Furthermore, the characterization of MAO knockout (KO) mice has revealed that the inactivation of this enzyme produces a number of functional and behavioral alterations, some of which may be harnessed for therapeutic aims. In this article, we discuss the intriguing hypothesis that the attenuation of the oxidative stress induced by the inactivation of either MAO isoform may contribute to both antidepressant and antiparkinsonian actions of MAO inhibitors. This possibility further highlights MAO inactivation as a rich source of novel avenues in the treatment of mental disorders.

An updated patent review: xanthine oxidase inhibitors for the treatment of hyperuricemia and gout (2011-2015).

PubMed

Ojha, Ritu; Singh, Jagjeet; Ojha, Anu; Singh, Harbinder; Sharma, Sahil; Nepali, Kunal

2017-03-01

Xanthine oxidase (XO) is a versatile molybdoflavoprotein, widely distributed, occurring in milk, kidney, lung, heart, and vascular endothelium. Catalysis by XO to produce uric acid and reactive oxygen species leads to many diseases. Anti hyperuricemic therapy by xanthine oxidase inhibitors has been mainly employed for the treatment of gout. Area covered: This review covers the patent literature (2011-2015) and also presents the interesting strategies/rational approaches employed for the design of xanthine oxidase inhibitors reported recently. Expert opinion: Recent literature indicates that various non purine scaffolds have been extensively investigated for xanthine oxidase inhibition. The significant potential endowed by heteroaryl based compounds, in particularly fused heterocycles clearly highlights their clinical promise and the need for detailed investigation. Studies by various research groups have also revealed that the flavone framework is open for isosteric replacements and structural modifications for yielding potent non purine xanthine oxidase inhibitors. In addition, various plant extracts recently reported to possess significant xanthine oxidase inhibitory potential presents enough promise to initiate a screening program for the identification of other plant extracts and phytoconstituents possessing inhibitory potential towards the enzyme.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

PubMed Central

Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh

2010-01-01

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker.

PubMed

Song, Zhongju; Yuan, Ruo; Chai, Yaqin; Jiang, Wen; Su, Huilan; Che, Xin; Ran, Xiaoqi

2011-01-15

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of glucose biosensor based on reconstitution of glucose oxidase onto polymeric redox mediator coated pencil graphite electrodes.

PubMed

Dervisevic, Muamer; Cevik, Emre; Senel, Mehmet

2015-01-01

In this study, a novel glucose biosensor was fabricated by reconstitutional immobilization of glucose oxidase (GOx) onto a poly(glycidyl methacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film coated pencil graphite electrode (PGE). The amperometric current response of poly(GMA-co-VFc)-GOx to glucose is linear in the concentration range between 1 and 16mM (correlation coefficient of 0.9998) with a detection limit of 2.7μM (S/N=3). Experimental parameters were studied in detail and optimized, including the pH and temperature governing the analytical performance of the biosensor. The stability and reusability of the biosensor as well as its kinetic parameters have also been studied. Copyright © 2014 Elsevier Inc. All rights reserved.

Genetics Home Reference: isolated sulfite oxidase deficiency

MedlinePlus

... Metabolic Disorders (CLIMB) March of Dimes: Amino Acid Metabolism Disorders The Compassionate Friends GeneReviews (1 link) Isolated Sulfite Oxidase Deficiency ClinicalTrials.gov (1 link) ClinicalTrials.gov Scientific Articles on PubMed (1 link) PubMed OMIM (1 link) ...

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102.

PubMed

El-Naggar, Noura El-Ahmady; Deraz, Sahar F; Soliman, Hoda M; El-Deeb, Nehal M; El-Shweihy, Nancy M

2017-03-29

There is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied. The best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 μg/mL. Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

Structural insights into electron transfer in caa3-type cytochrome oxidase

PubMed Central

Lyons, Joseph A.; Aragão, David; Slattery, Orla; Pisliakov, Andrei V.; Soulimane, Tewfik; Caffrey, Martin

2012-01-01

Summary Paragraph Cytochrome c oxidase is a member of the heme copper oxidase superfamily (HCO)1. HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme’s function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c2–4. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented. PMID:22763450

A mutation in a coproporphyrinogen III oxidase gene confers growth inhibition, enhanced powdery mildew resistance and powdery mildew-induced cell death in Arabidopsis.

PubMed

Guo, Chuan-yu; Wu, Guang-heng; Xing, Jin; Li, Wen-qi; Tang, Ding-zhong; Cui, Bai-ming

2013-05-01

A gene encoding a coproporphyrinogen III oxidase mediates disease resistance in plants by the salicylic acid pathway. A number of genes that regulate powdery mildew resistance have been identified in Arabidopsis, such as ENHANCED DISEASE RESISTANCE 1 to 3 (EDR1 to 3). To further study the molecular interactions between the powdery mildew pathogen and Arabidopsis, we isolated and characterized a mutant that exhibited enhanced resistance to powdery mildew. The mutant also showed dramatic powdery mildew-induced cell death as well as growth defects and early senescence in the absence of pathogens. We identified the affected gene by map-based cloning and found that the gene encodes a coproporphyrinogen III oxidase, a key enzyme in the tetrapyrrole biosynthesis pathway, previously known as LESION INITIATION 2 (LIN2). Therefore, we designated the mutant lin2-2. Further studies revealed that the lin2-2 mutant also displayed enhanced resistance to Hyaloperonospora arabidopsidis (H.a.) Noco2. Genetic analysis showed that the lin2-2-mediated disease resistance and spontaneous cell death were dependent on PHYTOALEXIN DEFICIENT 4 (PAD4), SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), which are all involved in salicylic acid signaling. Furthermore, the relative expression levels of defense-related genes were induced after powdery mildew infection in the lin2-2 mutant. These data indicated that LIN2 plays an important role in cell death control and defense responses in plants.

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

PubMed

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.

Monoamine oxidase B layer-by-layer film fabrication and characterization toward dopamine detection.

PubMed

Miyazaki, Celina Massumi; Pereira, Tamyris Paschoal; Mascagni, Daniela Branco Tavares; de Moraes, Marli Leite; Ferreira, Marystela

2016-01-01

In this work nanostructured film composites of the monoamine oxidase B (MAO-B) enzyme, free or encapsulated in liposomes, were fabricated by the layer-by-layer (LbL) self-assembly technique, employing polyethylene imine (PEI) as polycation. Initially, the MAO-B enzyme was incorporated into liposomes in order to preserve its enzymatic structure ensuring their activity and catalytic stability. The LbL film growth was monitored by surface plasmon resonance (SPR) by gold resonance angle shift analysis after each bilayer deposition. Subsequently, the films were applied as amperometric biosensors for dopamine detection using Prussian Blue (PB) as the electron mediator. The biosensor fabricated by MAO-B incorporated into liposomes composed of DPPG:POPG in the ratio (1:4) (w/w) showed the best performance with a sensitivity of 0.86 (μA cm(-2))/(mmol L(-1)) and a detection limit of 0.33 mmol L(-1).

Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase.

PubMed

Sahin, Huseyin

2016-01-01

The aim of this study was to evaluate new natural inhibitor sources for the enzymes urease and xanthine oxidase (XO). Chestnut, oak and polyfloral honey extracts were used to determine inhibition effects of both enzymes. In addition to investigate inhibition, the antioxidant capacities of these honeys were determined using total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity assays. Due to their high phenolic content, chestnut and oak honeys are found to be a powerful source for inhibition of both enzymes. Especially, oak honeys were efficient for urease inhibition with 0.012-0.021 g/mL IC50 values, and also chestnut honeys were powerful for XO inhibition with 0.028-0.039 g/mL IC50 values. Regular daily consumption of these honeys can prevent gastric ulcers deriving from Helicobacter pylori and pathological disorders mediated by reactive oxygen species.

Glucose oxidase immobilization on different modified surfaces of platinum nanowire for application in glucose detection

NASA Astrophysics Data System (ADS)

Thanh Tuyen Le, Thi; Duy Tran, Phu; Pham, Xuan Tung; Hien Tong, Duy; Chien Dang, Mau

2010-09-01

In this work, the surface of platinum (Pt) nanowires was modified by using several chemicals, including a compound of gelatin gel with SiO2, polyvinyl alcohol (PVA) with Prussian blue (PB) mediator and cysteamine self-assembled monolayers (SAM). Then, glucose oxidase (GOD) enzyme was immobilized on the modified surfaces of Pt nanowire electrodes by using techniques of electrochemical adsorption and chemical binding. The GOD immobilized Pt nanowires were used for application in glucose detection by performing a cyclic voltammetry measurement. The detection results showed that GOD was immobilized on all of the tested surfaces and the highest glucose detection sensitivity of 60 μM was obtained when the Pt nanowires were modified by PVA with PB mediator. Moreover, the sensors showed very high current response when the Pt nanowires were modified with the cysteamine SAM. The stability and catalyst activity of GOD are also reported here. For instance, the catalyst activity of GOD retained about 60% of its initial value after it was stored at 4 °C in a 100 mM PBS buffer solution with a pH of 7.2 for a period of 30 days.

Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heroux, A.; Bozinovski, D; Valley, M

2009-01-01

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 {angstrom} resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral nitroalkanes. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. Themore » oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066). The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.« less

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp.

PubMed

Van Hellemond, J J; Simons, B; Millenaar, F F; Tielens, A G

1998-01-01

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.

Androgen receptor and monoamine oxidase polymorphism in wild bonobos

PubMed Central

Garai, Cintia; Furuichi, Takeshi; Kawamoto, Yoshi; Ryu, Heungjin; Inoue-Murayama, Miho

2014-01-01

Androgen receptor gene (AR), monoamine oxidase A gene (MAOA) and monoamine oxidase B gene (MAOB) have been found to have associations with behavioral traits, such as aggressiveness, and disorders in humans. However, the extent to which similar genetic effects might influence the behavior of wild apes is unclear. We examined the loci AR glutamine repeat (ARQ), AR glycine repeat (ARG), MAOA intron 2 dinucleotide repeat (MAin2) and MAOB intron 2 dinucleotide repeat (MBin2) in 32 wild bonobos, Pan paniscus, and compared them with those of chimpanzees, Pan troglodytes, and humans. We found that bonobos were polymorphic on the four loci examined. Both loci MAin2 and MBin2 in bonobos showed a higher diversity than in chimpanzees. Because monoamine oxidase influences aggressiveness, the differences between the polymorphisms of MAin2 and MBin2 in bonobos and chimpanzees may be associated with the differences in aggression between the two species. In order to understand the evolution of these loci and AR, MAOA and MAOB in humans and non-human primates, it would be useful to conduct future studies focusing on the potential association between aggressiveness, and other personality traits, and polymorphisms documented in bonobos. PMID:25606465

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yinxi; Liu, Dan; Zhang, Huifeng

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 NADPHmore » 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

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

Treesearch

Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

2009-01-01

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase.

PubMed

Carro, Juan; Ferreira, Patricia; Martínez, Angel T; Gadda, Giovanni

2018-03-20

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α- 2 H 2 ]- p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s -1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H + transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H + from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O 2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.

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

PubMed

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.

Xanthine oxidase biosensor for monitoring meat spoilage

NASA Astrophysics Data System (ADS)

Vanegas, D. C.; Gomes, C.; McLamore, E. S.

2014-05-01

In this study, we have designed an electrochemical biosensor for real-time detection of specific biomarkers of bacterial metabolism related to meat spoilage (hypoxanthine and xanthine). The selective biosensor was developed by assembling a `sandwich' of nanomaterials and enzymes on a platinum-iridium electrode (1.6 mm tip diameter). The materials deposited on the sensor tip include amorphous platinum nanoclusters (i.e. Pt black), reduced graphene oxide, nanoceria, and xanthine oxidase. Xanthine oxidase was encapsulated in laponite hydrogel and used for the biorecognition of hypoxanthine and xanthine (two molecules involved in the rotting of meat by spoilage microorganisms). The developed biosensor demonstrated good electrochemical performance toward xanthine with sensitivity of 2.14 +/- 1.48 μA/mM, response time of 5.2 +/- 1.5 sec, lower detection limit of 150 +/- 39 nM, and retained at least 88% of its activity after 7 days of continuous use.

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

PubMed

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-03-16

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Immunological comparison of sulfite oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pollock, V.; Barber, M.J.

1991-03-11

Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibitedmore » S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.« less

Root respiratory burst oxidase homologue-dependent H2O2 production confers salt tolerance on a grafted cucumber by controlling Na+ exclusion and stomatal closure

PubMed Central

Niu, Mengliang; Huang, Yuan; Sun, Shitao; Sun, Jingyu; Cao, Haishun; Shabala, Sergey

2018-01-01

Abstract Plant salt tolerance can be improved by grafting onto salt-tolerant rootstocks. However, the underlying signaling mechanisms behind this phenomenon remain largely unknown. To address this issue, we used a range of physiological and molecular techniques to study responses of self-grafted and pumpkin-grafted cucumber plants exposed to 75 mM NaCl stress. Pumpkin grafting significantly increased the salt tolerance of cucumber plants, as revealed by higher plant dry weight, chlorophyll content and photochemical efficiency (Fv/Fm), and lower leaf Na+ content. Salinity stress resulted in a sharp increase in H2O2 production, reaching a peak 3 h after salt treatment in the pumpkin-grafted cucumber. This enhancement was accompanied by elevated relative expression of respiratory burst oxidase homologue (RBOH) genes RbohD and RbohF and a higher NADPH oxidase activity. However, this increase was much delayed in the self-grafted plants, and the difference between the two grafting combinations disappeared after 24 h. The decreased leaf Na+ content of pumpkin-grafted plants was achieved by higher Na+ exclusion in roots, which was driven by the Na+/H+ antiporter energized by the plasma membrane H+-ATPase, as evidenced by the higher plasma membrane H+-ATPase activity and higher transcript levels for PMA and SOS1. In addition, early stomatal closure was also observed in the pumpkin-grafted cucumber plants, reducing water loss and maintaining the plant’s hydration status. When pumpkin-grafted plants were pretreated with an NADPH oxidase inhibitor, diphenylene iodonium (DPI), the H2O2 level decreased significantly, to the level found in self-grafted plants, resulting in the loss of the salt tolerance. Inhibition of the NADPH oxidase-mediated H2O2 signaling in the root also abolished a rapid stomatal closure in the pumpkin-grafted plants. We concluded that the pumpkin-grafted cucumber plants increase their salt tolerance via a mechanism involving the root-sourced respiratory

Glutamate Excitotoxicity Linked to Spermine Oxidase Overexpression.

PubMed

Pietropaoli, Stefano; Leonetti, Alessia; Cervetto, Chiara; Venturini, Arianna; Mastrantonio, Roberta; Baroli, Giulia; Persichini, Tiziana; Colasanti, Marco; Maura, Guido; Marcoli, Manuela; Mariottini, Paolo; Cervelli, Manuela

2018-02-03

Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus. In Dach-SMOX astrocytes and neurons, an alteration of the phosphorylated and non-phosphorylated subunits of glutamate receptors increases the kainic acid response in these mice. Moreover, a decrease in excitatory amino acid transporters and an increase in the system x c - transporter, a Nrf-2 target, was observed. Sulfasalazine, a system x c - transporter inhibitor, was shown to revert the increased susceptibility of Dach-SMOX mice treated with kainic acid. We demonstrated that astrocytes play a crucial role in this process: neuronal spermine oxidase overexpression resulted in an alteration of glutamate excitability, in glutamate uptake and efflux in astrocytes involved in the synapse. Considering the involvement of oxidative stress in many neurodegenerative diseases, Dach-SMOX transgenic mouse can be considered as a suitable in vivo genetic model to study the involvement of spermine oxidase in excitotoxicity, which can be considered as a possible therapeutic target.

Design, synthesis and biological evaluation of novel xanthine oxidase inhibitors bearing a 2-arylbenzo[b]furan scaffold.

PubMed

Tang, Hong-Jin; Li, Wei; Zhou, Mei; Peng, Li-Ying; Wang, Jin-Xin; Li, Jia-Huang; Chen, Jun

2018-05-10

Xanthine oxidase, which catalyzes the oxidative reaction of hypoxanthine and xanthine into uric acid, is a key enzyme to the pathogenesis of hyperuricemia and gout. In this study, for the purpose of discovering novel xanthine oxidase (XO) inhibitors, a series of 2-arylbenzo[b]furan derivatives (3a-3d, 4a-4o and 6a-6d) were designed and synthesized. All these compounds were evaluated their xanthine oxidase inhibitory and antioxidant activities by using in vitro enzymatic assay and cellular model. The results showed that a majority of the designed compounds exhibited potent xanthine oxidase inhibitory effects and antioxidant activities, and compound 4a emerged as the most potent xanthine oxidase inhibitor (IC 50  = 4.45 μM). Steady-state kinetic measurements of the inhibitor 4a with the bovine milk xanthine oxidase indicated a mixed type inhibition with 3.52 μM K i and 13.14 μM K is , respectively. The structure-activity relationship analyses have also been presented. Compound 4a exhibited the potent hypouricemic effect in the potassium oxonate-induced hyperuricemic mice model. A molecular docking study of compound 4a was performed to gain an insight into its binding mode with xanthine oxidase. These results highlight the identification of a new class of xanthine oxidase inhibitors that have potential to be more efficacious in treatment of gout. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

PubMed Central

Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

2014-01-01

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-d-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. PMID:24488923

A decade of crystallization drops: crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri.

PubMed

Buschmann, Sabine; Richers, Sebastian; Ermler, Ulrich; Michel, Hartmut

2014-04-01

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality. © 2014 The Protein Society.

Age-related ultrastructural and monoamine oxidase changes in the rat optic nerve.

PubMed

Taurone, S; Ripandelli, G; Minni, A; Lattanzi, R; Miglietta, S; Pepe, N; Fumagalli, L; Micera, A; Pastore, F S; Artico, M

2016-01-01

The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

PubMed Central

Glerum, M; Robinson, B H; Spratt, C; Wilson, J; Patrick, D

1987-01-01

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient. PMID:2821802

Genetics Home Reference: cytochrome c oxidase deficiency

MedlinePlus

... are caused by mutations in genes found within nuclear DNA; however, in some rare instances, mutations in genes located within mtDNA cause this condition. The genes associated with cytochrome c oxidase deficiency are involved in energy production in mitochondria through a process called oxidative ...

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

PubMed

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.

NADPH Oxidase Inhibition Improves Neurological Outcomes in Surgically-Induced Brain Injury

PubMed Central

Lo, Wendy; Bravo, Thomas; Jadhav, Vikram; Zhang, John H.; Tang, Jiping

2007-01-01

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit of NADPH oxidase (gp91phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 hours post-SBI in gp91phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5mg/kg, i.p. 30 minutes before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 hours post SBI. The gp91phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 hours post-SBI by apocynin pretreatment but not at 24 hours post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI. PMID:17317004

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum

Treesearch

Diane Dietrich; Casey Crooks

2009-01-01

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5’UTR...

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

PubMed

Xu, Y L; Li, L; Wu, K; Peeters, A J; Gage, D A; Zeevaart, J A

1995-07-03

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Polyamine oxidase activity in rats treated with mitoguazone: specific and permanent decrease in thymus.

PubMed

Ferioli, M E; Armanni, A

2003-01-01

To extend the knowledge on the role of polyamine oxidase in thymus physiology, we evaluated the in vivo effect of the polyamine biosynthetic pathway inhibitor mitoguazone. The drug markedly and permanently decreased the enzyme activity in the organ, in which the level of putrescine also decreased at the later times observed. A byproduct of the reaction catalyzed by polyamine oxidase is hydrogen peroxide, a well known inducer of apoptosis. The decrease in polyamine oxidase activity, with the consequent decrease in hydrogen peroxide production, is correlated with a positive effect on thymus physiology. Since mitoguazone has been successfully employed in patients with AIDS-related diseases, in which the reconstitution of the immune function is a favorable prognostic index, we hypothesized that mitoguazone may have the thymus as target organ, and that the decrease in polyamine oxidase activity may have a role in the positive effect of the drug.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

PubMed Central

Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

2012-01-01

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis

PubMed Central

Ruiz, Lynnette A.; Báez-Vega, Perla M.; Ruiz, Abigail; Peterse, Daniëlle P.; Monteiro, Janice B.; Bracero, Nabal; Beauchamp, Pedro; Fazleabas, Asgerally T.; Flores, Idhaliz

2015-01-01

Lysyl oxidases (LOXs) are enzymes involved in collagen deposition, extracellular membrane remodeling, and invasive/metastatic potential. Previous studies reveal an association of LOXs and endometriosis. We aimed to identify the mechanisms activated by upregulation of lysyl oxidases (LOX) in endometriotic cells and tissues. We hypothesized that LOX plays a role in endometriosis by promoting invasiveness and epithelial to mesenchymal transition (EMT). Methods: The LOX protein expression levels were measured by immunohistochemistry in lesions and endometrium on a tissue microarray (TMA) and in endometrial biopsies from patients and controls during the window of implantation (WOI). Estradiol regulation of LOX expression was determined by quantitative polymerase chain reaction (qPCR). Proliferation, invasion, and migration assays were performed in epithelial (endometrial epithelial cell), endometrial (human endometrial stromal cell), and endometriotic cell lines (ECL and 12Z). Pathway-focused multiplex qPCR was used to determine transcriptome changes due to LOX overexpression. Results: LOX protein was differentially expressed in ovarian versus peritoneal lesions. During WOI, LOX levels were higher in luminal epithelium of patients with endometriosis-associated infertility compared to controls. Invasive epithelial cell lines expressed higher levels of LOX than noninvasive ones. Transfection of LOX into noninvasive epithelial cells increased their migration in an LOX inhibitor-sensitive manner. Overexpression of LOX did not fully induce EMT but the expression of genes related to fibrosis and extracellular matrix remodeling were dysregulated. Conclusions: This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions

Role of Valine 464 in the Flavin Oxidation Reaction Catalyzed by Choline Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finnegan, Steffan; Agniswamy, Johnson; Weber, Irene T.

2010-11-03

The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants {ge} 10{sup 5} M{sup -1} s{sup -1} and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 {angstrom}more » from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.« less

Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity.

PubMed

Niu, Yanfen; Liu, Jia; Liu, Hai-Yang; Gao, Li-Hui; Feng, Guo-Hua; Liu, Xu; Li, Ling

2016-09-01

Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action. Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3-9) toward xanthine oxidase was also evaluated. Materials and methods For a dose-dependent study, mangiferin (1.5-6.0 mg/kg) and norathyriol (0.92-3.7 mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1 μmol/kg. In vitro, inhibition of test compounds (2.4-2.4 mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver-Burk plots. Results Norathyriol (0.92, 1.85 and 3.7 mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC50 value of 44.6 μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver-Burk plots. The structure-activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition. Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

PubMed Central

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

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

PubMed Central

Suh, Sang Won; Gum, Elizabeth T.; Hamby, Aaron M.; Chan, Pak H.; Swanson, Raymond A.

2007-01-01

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase. PMID:17404617

Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction Phenotype in Arabidopsis

PubMed Central

Huang, Shihshieh; Raman, Anuradha S.; Ream, Joel E.; Fujiwara, Hideji; Cerny, R. Eric; Brown, Sherri M.

1998-01-01

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C19-GAs. All bioactive GAs are C19-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA1, GA9, and GA20 were detected in seedlings of the transgenic line examined. GA4, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype. PMID:9808721

Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

PubMed

Moosavi-Nejad, S Zahra; Moosavi-Movahedi, Ali-Akbar; Rezaei-Tavirani, Mostafa; Floris, Giovanni; Medda, Rosaria

2003-03-31

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline.

PubMed

Berney, Michael; Hartman, Travis E; Jacobs, William R

2014-07-15

The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. Importance: A major drawback of current TB chemotherapy is its long duration. New drug regimens with rapid killing kinetics are desperately needed. Our study demonstrates that inhibition of a nonessential bacterial enzyme greatly improves the efficacy of the latest TB drug bedaquiline and emphasizes that screening for compounds with synergistic killing mechanisms is a promising strategy. Copyright © 2014 Berney et al.

The glucose oxidase-peroxidase assay for glucose

USDA-ARS?s Scientific Manuscript database

The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.

PubMed

Ferrari, Alessandro R; Lee, Misun; Fraaije, Marco W

2015-06-01

Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature. © 2015 Wiley Periodicals, Inc.

Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components

PubMed Central

Morgan, Deri; Cherny, Vladimir V.; Price, Marianne O.; Dinauer, Mary C.; DeCoursey, Thomas E.

2002-01-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O2 −) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H+ efflux was thought to be contained within the gp91phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063–36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COSphox). The 7D5 antibody, which detects an extracellular epitope of the gp91phox protein, labeled 96–98% of COSphox cells. NADPH oxidase was functional because COSphox (but not COSWT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COSWT) or COSphox cells studied at pHo 7.0 and pHi 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H+ current in COSWT or COSphox cells. Therefore, gp91phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase. PMID:12034764

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

PubMed

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.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet

PubMed Central

Amaral-de-Carvalho, Diogo; Oliveira, Elsa; Alves, Ângela; Costa, Vítor; Calado, Gonçalo

2018-01-01

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet. PMID:29529078

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

PubMed Central

Kalia, Nitin P.; Hasenoehrl, Erik J.; Ab Rahman, Nurlilah B.; Koh, Vanessa H.; Ang, Michelle L. T.; Sajorda, Dannah R.; Hards, Kiel; Grüber, Gerhard; Alonso, Sylvie; Cook, Gregory M.; Berney, Michael; Pethe, Kevin

2017-01-01

The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis. PMID:28652330

Peroxynitrite mediates testosterone-induced vasodilation of microvascular resistance vessels.

PubMed

Puttabyatappa, Yashoda; Stallone, John N; Ergul, Adviye; El-Remessy, Azza B; Kumar, Sanjiv; Black, Stephen; Johnson, Maribeth; Owen, Mary P; White, Richard E

2013-04-01

Our knowledge of how androgens influence the cardiovascular system is far from complete, and this lack of understanding is especially true of how androgens affect resistance vessels. Our aim was to identify the signaling mechanisms stimulated by testosterone (TES) in microvascular arteries and to understand how these mechanisms mediate TES-induced vasodilation. Mesenteric microvessels were isolated from male Sprague-Dawley rats. Tension studies demonstrated a rapid, concentration-dependent, vasodilatory response to TES that did not involve protein synthesis or aromatization to 17β-estradiol. Dichlorofluorescein fluorescence and nitrotyrosine immunoblot experiments indicated that TES stimulated peroxynitrite formation in microvessels, and functional studies demonstrated that TES-induced vasodilation was inhibited by scavenging peroxynitrite. As predicted, TES enhanced the production of both peroxynitrite precursors (i.e., superoxide and nitic oxide), and xanthine oxidase was identified as the likely source of TES-stimulated superoxide production. Functional and biochemical studies indicated that TES signaling involved activity of the phosphoinositide 3 (PI3) kinase-protein kinase B (Akt) cascade initiated by activation of the androgen receptor and culminated in enhanced production of cGMP and microvascular vasodilation. These findings, derived from a variety of analytical and functional approaches, provide evidence for a novel nongenomic signaling mechanism for androgen action in the microvasculature: TES-stimulated vasodilation mediated primarily by peroxynitrite formed from xanthine oxidase-generated superoxide and NO. This response was associated with activation of the PI3 kinase-Akt signaling cascade initiated by activation of the androgen receptor. We propose this mechanism could account for TES-stimulated cGMP production in microvessels and, ultimately, vasodilation.

Peroxynitrite Mediates Testosterone-Induced Vasodilation of Microvascular Resistance Vessels

PubMed Central

Puttabyatappa, Yashoda; Stallone, John N.; Ergul, Adviye; El-Remessy, Azza B.; Kumar, Sanjiv; Black, Stephen; Johnson, Maribeth; Owen, Mary P.

2013-01-01

Our knowledge of how androgens influence the cardiovascular system is far from complete, and this lack of understanding is especially true of how androgens affect resistance vessels. Our aim was to identify the signaling mechanisms stimulated by testosterone (TES) in microvascular arteries and to understand how these mechanisms mediate TES-induced vasodilation. Mesenteric microvessels were isolated from male Sprague-Dawley rats. Tension studies demonstrated a rapid, concentration-dependent, vasodilatory response to TES that did not involve protein synthesis or aromatization to 17β-estradiol. Dichlorofluorescein fluorescence and nitrotyrosine immunoblot experiments indicated that TES stimulated peroxynitrite formation in microvessels, and functional studies demonstrated that TES-induced vasodilation was inhibited by scavenging peroxynitrite. As predicted, TES enhanced the production of both peroxynitrite precursors (i.e., superoxide and nitic oxide), and xanthine oxidase was identified as the likely source of TES-stimulated superoxide production. Functional and biochemical studies indicated that TES signaling involved activity of the phosphoinositide 3 (PI3) kinase-protein kinase B (Akt) cascade initiated by activation of the androgen receptor and culminated in enhanced production of cGMP and microvascular vasodilation. These findings, derived from a variety of analytical and functional approaches, provide evidence for a novel nongenomic signaling mechanism for androgen action in the microvasculature: TES-stimulated vasodilation mediated primarily by peroxynitrite formed from xanthine oxidase-generated superoxide and NO. This response was associated with activation of the PI3 kinase-Akt signaling cascade initiated by activation of the androgen receptor. We propose this mechanism could account for TES-stimulated cGMP production in microvessels and, ultimately, vasodilation. PMID:23318471

The Intimate and Controversial Relationship between Voltage Gated Proton Channels and the Phagocyte NADPH Oxidase

PubMed Central

DeCoursey, Thomas E.

2016-01-01

Summary One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the NADPH oxidase complex and voltage gated proton channels (HV1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987–1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV1, and HV1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase -- an industrial strength producer of reactive oxygen species (ROS) -- to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. PMID:27558336

The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

PubMed

DeCoursey, Thomas E

2016-09-01

One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

PubMed Central

Niki, Tomoya; Nishijima, Takaaki; Nakayama, Masayoshi; Hisamatsu, Tamotsu; Oyama-Okubo, Naomi; Yamazaki, Hiroko; Hedden, Peter; Lange, Theo; Mander, Lewis N.; Koshioka, Masaji

2001-01-01

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2–35S-Ω). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T2 generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T2 generation, indicating that the transgene was stable and dominant. The endogenous levels of GA1 and GA4 were reduced in the dwarfs, whereas large amounts of GA17 and GA25, which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants.

PubMed

Radi, Abeer; Lange, Theo; Niki, Tomoya; Koshioka, Masaji; Lange, Maria João Pimenta

2006-02-01

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

PubMed Central

Vujcic, Slavoljub; Diegelman, Paula; Bacchi, Cyrus J; Kramer, Debora L; Porter, Carl W

2002-01-01

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors. PMID:12141946

Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases.

PubMed

Wang, Huihui; Liu, Baobao; Li, Hongyan; Zhang, Shicui

2016-01-10

Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutation of the NADH Oxidase Gene (nox) Reveals an Overlap of the Oxygen- and Acid-Mediated Stress Responses in Streptococcus mutans

PubMed Central

Derr, Adam M.; Faustoferri, Roberta C.; Betzenhauser, Matthew J.; Gonzalez, Kaisha; Marquis, Robert E.

2012-01-01

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929–936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites. PMID:22179247

Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

PubMed Central

Rhoads, DM; McIntosh, L

1992-01-01

Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development

USDA-ARS?s Scientific Manuscript database

The mitochondrial alternative oxidase (AOX) is a non-energy conserving ubiquinol oxidase found in most fungal genomes studied to date. With the development of fungicides containing cytochrome-dependent respiratory chain (CRC) inhibitors, a strong interest in studying AOX functions in phytopathogenic...

Reduction of NADPH-Oxidase Activity Ameliorates the Cardiovascular Phenotype in a Mouse Model of Williams-Beuren Syndrome

PubMed Central

Campuzano, Victoria; Segura-Puimedon, Maria; Terrado, Verena; Sánchez-Rodríguez, Carolina; Coustets, Mathilde; Menacho-Márquez, Mauricio; Nevado, Julián; Bustelo, Xosé R.; Francke, Uta; Pérez-Jurado, Luis A.

2012-01-01

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. PMID:22319452

Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments.

PubMed

Zhou, Guangqi; Yin, Jianhua; Chen, Haijiang; Hua, Yijie; Sun, Linlin; Gao, Haichun

2013-09-01

Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.

Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42.

PubMed

El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M; El-Ewasy, Sara M

2016-09-20

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. 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 strain NEAE-42. The optimization of different process parameters for cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables were screened using Plackett-Burman experimental design. Cholesterol, initial pH and (NH4)2SO4 were the most significant positive independent variables affecting cholesterol oxidase production. Central composite design was chosen to elucidate the optimal concentrations of the selected process variables on cholesterol oxidase production. It was found that, cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 after optimization process was 20.521U/mL which is higher than result obtained from the basal medium before screening process using Plackett-Burman (3.31 U/mL) with a fold of increase 6.19. The cholesterol oxidase level production obtained in this study (20.521U/mL) by the statistical method is higher than many of the reported values.

IRON REGULATES XANTHINE OXIDASE ACTIVITY IN THE LUNG

EPA Science Inventory

The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreduct...

Molecular mechanisms of hypertension: role of Nox family NADPH oxidases.

PubMed

Sedeek, Mona; Hébert, Richard L; Kennedy, Chris R; Burns, Kevin D; Touyz, Rhian M

2009-03-01

Molecular mechanisms contributing to the pathoetiology of hypertension are complex, involving many interacting systems such as signaling through G protein-coupled receptors, the renin-angiotensin system, vascular inflammation and remodeling, vascular senescence and aging and developmental programming, as highlighted in the current issue of the journal. Common to these systems is NADPH oxidase-derived reactive oxygen species (ROS). This editorial highlights current concepts relating to the production of ROS in hypertension and focuses on the Nox family NADPH oxidases, major sources of free radicals in the cardiovascular and renal systems. ROS play a major role as intracellular signaling molecules to regulate normal biological cellular responses. In pathological conditions, loss of redox homeostasis contributes to vascular oxidative damage. Recent evidence indicates that specific enzymes, the Nox family of NADPH oxidases, have the sole function of generating ROS in a highly regulated fashion in physiological conditions, and that in disease states, hyperactivation of Noxes contributes to oxidative stress and consequent cardiovascular and renal injury. The Nox family comprises seven members, Nox1-Nox7. Nox1, Nox2 (gp91phox-containing NADPH oxidase), Nox4 and Nox5 have been identified in the cardiovascular-renal systems and have been implicated in the pathophysiology of cardiovascular and renal disease. Noxes, which are differentially regulated in hypertension, are major sources of cardiovascular and renal oxidative stress. This has evoked considerable interest because of the possibilities that therapies targeted against specific Nox isoforms to decrease ROS generation or to increase nitric oxide availability or both may be useful in minimizing vascular injury and renal dysfunction, and thereby prevent or regress target organ damage associated with hypertension.

Substitutions of S101 decrease proton and hydride transfers in the oxidation of betaine aldehyde by choline oxidase.

PubMed

Gadda, Giovanni; Yuan, Hongling

2017-11-15

Choline oxidase oxidizes choline to glycine betaine, with two flavin-mediated reactions to convert the alcohol substrate to the carbon acid product. Proton abstraction from choline or hydrated betaine aldehyde in the wild-type enzyme occurs in the mixing time of the stopped-flow spectrophotometer, thereby precluding a mechanistic investigation. Mutagenesis of S101 rendered the proton transfer reaction amenable to study. Here, we have investigated the aldehyde oxidation reaction catalyzed by the mutant enzymes using steady-state and rapid kinetics with betaine aldehyde. Stopped-flow traces for the reductive half-reaction of the S101T/V/C variants were biphasic, corresponding to the reactions of proton abstraction and hydride transfer. In contrast, the S101A enzyme yielded monophasic traces like wild-type choline oxidase. The rate constants for proton transfer in the S101T/C/V variants decreased logarithmically with increasing hydrophobicity of residue 101, indicating a behavior different from that seen previously with choline for which no correlation was determined. The rate constants for hydride transfer also showed a logarithmic decrease with increasing hydrophobicity at position 101, which was similar to previous results with choline as a substrate for the enzyme. Thus, the hydrophilic character of S101 is necessary not only for efficient hydride transfer but also for the proton abstraction reaction. Copyright © 2017. Published by Elsevier Inc.

Growth hormone and drug metabolism. Acute effects on microsomal mixed-function oxidase activities in rat liver.

PubMed Central

Wilson, J T; Spelsberg, T C

1976-01-01

Adult male rats were subjected either to sham operation or to hypophysectomy and adrenalectomy and maintained for a total of 10 days before treatment with growth hormone. Results of the early effects of growth hormone on the activities of the mixed-function oxidases in rat liver over a 96h period after growth-hormone treatment are presented. 2. Hypophysectomy and adrenalectomy result in decreased body and liver weight and decreased drug metabolism (mixed-function oxidases). Concentrations of electron-transport-system components are also decreased. 3. In the hypophysectomized/adrenalectomized rats, growth hormone decreases the activities of the liver mixed-function oxidases and the cytochrome P-450 and cytochrome c reductases, as well as decreasing the concentration of cytochrome P-450 compared with that of control rats. Similar but less dramatic results are obtained with sham-operated rats. 4. It is concluded that whereas growth hormone enhances liver growth, including induction of many enzyme activities, it results in a decrease in mixed-function oxidase activity. Apparently, mixed-function oxidase activity decreases in liver when growth (mitogenesis) increases. PMID:938458

Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

PubMed

Haruta, M; Murata, M; Kadokura, H; Homma, S

1999-03-01

An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

A multicopper oxidase contributes to the copper tolerance of Brucella melitensis 16M.

PubMed

Wu, Tonglei; Wang, Shaohua; Wang, Zhen; Peng, Xiaowei; Lu, Yanli; Wu, Qingmin

2015-06-01

Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-09-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

PubMed

Morgan, Deri; Cherny, Vladimir V; Price, Marianne O; Dinauer, Mary C; DeCoursey, Thomas E

2002-06-01

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

Isolation and characterization of a monoamine oxidase B selective inhibitor from tobacco smoke.

PubMed

Khalil, Ashraf A; Davies, Bruce; Castagnoli, Neal

2006-05-15

It is well established that tobacco smokers have reduced levels of monoamine oxidase activities both in the brain and peripheral organs. Furthermore, extensive evidence suggests that smokers are less prone to develop Parkinson's disease. These facts, plus the observation that inhibition of monoamine oxidase B protects against the parkinsonian inducing effects of the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, have prompted studies to identify monoamine oxidase inhibitors in the tobacco plant and tobacco cigarette smoke. Our previous efforts on cured tobacco leaf extracts have led to the characterization of 2,3,6-trimethyl-1,4-naphthoquinone, a non-selective monoamine oxidase inhibitor, and farnesylacetone, a selective monoamine oxidase B inhibitor. We now have extended these studies to tobacco smoke constituents. Fractionation of the smoke extracts has confirmed and extended the qualitative results of an earlier report [J. Korean Soc. Tob. Sci.1997, 19, 136] demonstrating the inhibitory activity of the terpene trans,trans-farnesol on rat brain MAO-B. In the present study, K(i) values for the inhibition of human, baboon, monkey, dog, rat, and mouse liver MAO-B have been determined. Noteworthy is the absence of inhibitory effects on human placental MAO-A and beef liver MAO-B. A limited structure-activity relationship study of analogs of trans,trans-farnesol is reported. Although the health hazards associated with the use of tobacco products preclude any therapeutic opportunities linked to smoking, these results suggest the possibility of identifying novel structures of compounds that could lead to the development of neuroprotective agents.

Recovery of choline oxidase activity by in vitro recombination of individual segments.

PubMed

Heinze, Birgit; Hoven, Nina; O'Connell, Timothy; Maurer, Karl-Heinz; Bartsch, Sebastian; Bornscheuer, Uwe T

2008-11-01

Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37 degrees C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.

Longevity and aging. Role of free radicals and xanthine oxidase. A review.

PubMed

Labat-Robert, J; Robert, L

2014-04-01

Longevity and aging are differently regulated. Longevity has an important part of genetic determinants, aging is essentially post-genetic. Among the genes involved in longevity determination, sirtuins, activated also by calorie restriction and some others as the TOR pathway, attracted special interest after the insulin–IGF pathway first shown to regulate longevity in model organisms. For most of these genes, postponement of life-threatening diseases is the basis of their action which never exceeds about 35% of all determinants, in humans. Among the post-genetic mechanisms responsible for age-related decline of function, free radicals attracted early interest as well as the Maillard reaction, generating also free radicals. Most attempts to remediate to free radical damage failed however, although different scavenger mechanisms and protective substances are present in the organism. Synthetic protectors were also tested without success. The only example of a successful treatment of a free radical mediated pathology is the case of xanthine oxidase, involved in cardiovascular pathology, essentially during the ischemia-reperfusion process. Its inhibition by allopurinol is currently used to fight this deadly syndrome.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

PubMed

El Bakkali-Tahéri, Nadia; Tachon, Sybille; Orio, Maylis; Bertaina, Sylvain; Martinho, Marlène; Robert, Viviane; Réglier, Marius; Tron, Thierry; Dorlet, Pierre; Simaan, A Jalila

2017-06-01

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of the nitric oxide oxidase activity of myeloperoxidase by pharmacological agents.

PubMed

Maiocchi, Sophie L; Morris, Jonathan C; Rees, Martin D; Thomas, Shane R

2017-07-01

The leukocyte-derived heme enzyme myeloperoxidase (MPO) is released extracellularly during inflammation and impairs nitric oxide (NO) bioavailability by directly oxidizing NO or producing NO-consuming substrate radicals. Here, structurally diverse pharmacological agents with activities as MPO substrates/inhibitors or antioxidants were screened for their effects on MPO NO oxidase activity in human plasma and physiological model systems containing endogenous MPO substrates/antioxidants (tyrosine, urate, ascorbate). Hydrazide-based irreversible/reversible MPO inhibitors (4-ABAH, isoniazid) or the sickle cell anaemia drug, hydroxyurea, all promoted MPO NO oxidase activity. This involved the capacity of NO to antagonize MPO inhibition by hydrazide-derived radicals and/or the ability of drug-derived radicals to stimulate MPO turnover thereby increasing NO consumption by MPO redox intermediates or NO-consuming radicals. In contrast, the mechanism-based irreversible MPO inhibitor 2-thioxanthine, potently inhibited MPO turnover and NO consumption. Although the phenolics acetaminophen and resveratrol initially increased MPO turnover and NO consumption, they limited the overall extent of NO loss by rapidly depleting H 2 O 2 and promoting the formation of ascorbyl radicals, which inefficiently consume NO. The vitamin E analogue trolox inhibited MPO NO oxidase activity in ascorbate-depleted fluids by scavenging NO-consuming tyrosyl and urate radicals. Tempol and related nitroxides decreased NO consumption in ascorbate-replete fluids by scavenging MPO-derived ascorbyl radicals. Indoles or apocynin yielded marginal effects. Kinetic analyses rationalized differences in drug activities and identified criteria for the improved inhibition of MPO NO oxidase activity. This study reveals that widely used agents have important implications for MPO NO oxidase activity under physiological conditions, highlighting new pharmacological strategies for preserving NO bioavailability during

Spectrophotometric determination of H2O2-generating oxidases using oxyhemoglobin as oxygen donor and indicator.

PubMed

Bârzu, O; Dânşoreanu, M

1980-01-01

1. Spectrophotometric determination of oxygen uptake using oxyhemoglobin as oxygen donor and indicator was used for assay of H2O2-generating oxidases like monoamine oxidase and glucose oxidase. 2. In order to decompose H2O2 formed during the oxygen uptake, catalase and methanol (or ethanol) was added to the respiratory system. At pH values higher than 7.5 the oxydation of deoxygenated hemoglobin to methemoglobin was less than 3%. 2. Oxidases with low Km for oxygen can be assayed using the spectrophotometric method if suitable correction factors are introduced into the calculation of oxygen uptake. The correction factor represents the ratio of the rate of formation (or disappearance) of one of the reactants and the rate of oxyhemoglobin deoxygenation, measured under identical experimental conditions.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

NASA Astrophysics Data System (ADS)

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.

Exercise Training, NADPH Oxidase p22phox Gene Polymorphisms, and Hypertension

PubMed Central

FEAIRHELLER, DEBORAH L.; BROWN, MICHAEL D.; PARK, JOON-YOUNG; BRINKLEY, TINA E.; BASU, SAMAR; HAGBERG, JAMES M.; FERRELL, ROBERT E.; FENTY-STEWART, NICOLA M.

2010-01-01

Introduction Oxidative stress that is mediated through NADPH oxidase activity plays a role in the pathology of hypertension, and aerobic exercise training reduces NADPH oxidase activity. The involvement of genetic variation in the p22phox (CYBA) subunit genes in individual oxidative stress responses to aerobic exercise training has yet to be examined in Pre and Stage 1 hypertensives. Methods Ninety-four sedentary Pre and Stage 1 hypertensive adults underwent 6 months of aerobic exercise training at a level of 70% V̇O2max to determine whether the CYBA polymorphisms, C242T and A640G, were associated with changes in urinary 8-iso-prostaglandin F2α (8-iso-PGF2α), urinary nitric oxide metabolites (NOx), and plasma total antioxidant capacity (TAC). Results Demographic and subject characteristics were similar among genotype groups for both polymorphisms. At baseline, a significant (P = 0.03) difference among the C2424T genotype groups in 8-iso-PGF2α levels was detected, with the TT homozygotes having the lowest levels and the CC homozygotes having the highest levels. However, no differences were found at baseline between the A640G genotype groups. After 6 months of aerobic exercise training, there was a significant increase in V̇O2max (P < 0.0001) in the entire study population. In addition, there were significant increases in both urinary 8-iso-PGF2α (P = 0.002) and plasma TAC (P = 0.03) levels and a significant decrease in endogenous urinary NOx (P < 0.0001). Overall, aerobic exercise training elicited no significant differences among genotype groups in either CYBA variant for any of the oxidative stress variables. Conclusions We found that compared with CYBA polymorphisms C242T and A640G, it was aerobic exercise training that had the greatest influence on the selected biomarkers; furthermore, our results suggest that the C242T CYBA variant influences baseline levels of urinary 8-iso-PGF2α but not the aerobic exercise-induced responses. PMID:19516159

Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

PubMed

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.

The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction.

PubMed

Meneshian, Avedis; Bulkley, Gregory B

2002-07-01

Xanthine oxidoreductase (XOR) is a ubiquitous metalloflavoprotein that appears in two interconvertible yet functionally distinct forms: xanthine dehydrogenase (XD), which is constitutively expressed in vivo; and xanthine oxidase (XO), which is generated by the posttranslational modification of XD, either through the reversible, incremental thiol oxidation of sulfhydryl residues on XD or the irreversible proteolytic cleavage of a segment of XD, which occurs at low oxygen tension and in the presence of several proinflammatory mediators. Functionally, both XD and XO catalyze the oxidation of purines to urate. However, whereas XD requires NAD+ as an electron acceptor for these redox reactions, thereby generating the stable product NADH, XO is unable to use NAD+ as an electron acceptor, requiring instead the reduction of molecular oxygen for this purine oxidation and generating the highly reactive superoxide free radical. Nearly 100 years of study has documented the physiologic role of XD in urate catabolism. However, the rapid, posttranslational conversion of XD to the oxidant-generating form XO provides a possible physiologic mechanism for rapid, posttranslational, oxidant-mediated signaling. XO-generated reactive oxygen species (ROS) have been implicated in various clinicopathologic entities, including ischemia/reperfusion injury and multisystem organ failure. More recently, the concept of physiologic signal transduction mediated by ROS has been proposed, and the possibility of XD to XO conversion, with subsequent ROS generation, serving as the trigger of the microvascular inflammatory response in vivo has been hypothesized. This review presents the evidence and basis for this hypothesis.

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori.

PubMed

Gouka, R J; van der Heiden, M; Swarthoff, T; Verrips, C T

2001-06-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.

Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

PubMed Central

Gouka, Robin J.; van der Heiden, Monique; Swarthoff, Ton; Verrips, C. Theo

2001-01-01

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning. PMID:11375170

Reductive trapping of substrate to bovine plasma amine oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartmann, C.; Klinman, J.P.

1987-01-25

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O/sub 2/ to H/sub 2/O/sub 2/. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation betweenmore » a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of (/sup 14/C)benzylamine and (/sup 3/H)NaCNBH/sub 3/. The use of the relatively weak reductant, NaCNBH/sub 3/, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH/sub 3/ in the presence of substrate, leading to the incorporation of 1 mol of (/sup 14/C)benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with (/sup 3/H)NaCNBH/sub 3/, analogous to an earlier study with (/sup 3/H)NaCNBH/sub 4/ (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.« less

The role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes in the [omim][BF4]-mediated toxic mode of action in mussel hemocytes.

PubMed

Belavgeni, Alexia; Dailianis, Stefanos

2017-09-01

The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF 4 ])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF 4 ] (0.1-10 mg L -1 ) and thereafter [omim][BF 4 ]-mediated oxidative (in terms of superoxide anions/O 2 - and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L -1 [omim][BF 4 ]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF 4 ]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 μM) and N G -nitro- l -arginine methyl ester (l-NAME 10 μM), respectively. The results showed that [omim][BF 4 ] ability to enhance O 2 - , NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF 4 ]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition

PubMed Central

Yoon, Bo-Eun; Woo, Junsung; Chun, Ye-Eun; Chun, Heejung; Jo, Seonmi; Bae, Jin Young; An, Heeyoung; Min, Joo Ok; Oh, Soo-Jin; Han, Kyung-Seok; Kim, Hye Yun; Kim, Taekeun; Kim, Young Soo; Bae, Yong Chul; Lee, C Justin

2014-01-01

GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5–10 mm by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca2+-induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain. PMID:25239459

An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

PubMed Central

Jo, Jeanyoung; Cortez, Krista L; Cornell, William Cole; Price-Whelan, Alexa

2017-01-01

Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets. PMID:29160206

Utilization of Diamine Oxidase Enzyme from Mung Bean Sprouts (Vigna radiata L) for Histamine biosensors

NASA Astrophysics Data System (ADS)

Karim, Abdul; Wahab, A. W.; Raya, I.; Natsir, H.; Arif, A. R.

2018-03-01

This research is aimed to utilize the diamine oxidase enzyme (DAO) which isolated from mung bean sprouts (Vigna radiata L) to develop histamine biosensors based on electode enzyme with the amperometric method (cyclic voltammetry).The DAO enzyme is trapped inside the membrane of chitin-cellulose acetate 2:1 and glutaraldehyde which super imposed on a Pt electrode. Histamine will be oxidized by DAO enzyme to produce aldehydes and H2O2 that acting as electron transfer mediators.The performance of biosensors will be measured at various concentrations of glutaraldehyde, temperature changes and different range of pH. Recently, it has been found that the optimal conditions obtained from the paramaters as follows; at 25% of glutaraldehyde, temperature of 37°C and pH of 7.4. Eventually, the results provided an expectation for applying histamine biosensors in determining the freshness and safety of fish specifically skombroidae families.

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

PubMed

Altenhöfer, Sebastian; Radermacher, Kim A; Kleikers, Pamela W M; Wingler, Kirstin; Schmidt, Harald H H W

2015-08-10

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

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

PubMed

Zafred, Domen; Steiner, Barbara; Teufelberger, Andrea R; Hromic, Altijana; Karplus, P Andrew; Schofield, Christopher J; Wallner, Silvia; Macheroux, Peter

2015-08-01

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF. © 2015 FEBS.

Characterization of two brassinosteroid C-6 oxidase genes in pea.

PubMed

Jager, Corinne E; Symons, Gregory M; Nomura, Takahito; Yamada, Yumiko; Smith, Jennifer J; Yamaguchi, Shinjiro; Kamiya, Yuji; Weller, James L; Yokota, Takao; Reid, James B

2007-04-01

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yun-Ling; Li, Li; Wu, Keqiang

1995-07-03

The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidasemore » gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6

Purification and properties of two terminal oxidase complexes of Escherichia coli aerobic respiratory chain.

PubMed

Kita, K; Konishi, K; Anraku, Y

1986-01-01

Two terminal oxidase complexes, cytochrome b-562-o complex and cytochrome b-558-d complex, are isolated in highly purified forms which show ubiquinol oxidase activities. From the result of steady-state kinetics of cytochromes in the membrane and E'm values of purified cytochromes, we propose a branched arrangement of the late exponential phase of aerobic growth, as shown in Fig. 10. Cytochrome b-556 is reduced by several dehydrogenases and the gene for this cytochrome (cybA) is located in the sdh gene cluster. Recently, we found another low-potential b-type cytochrome, cytochrome b-561 (Em' = 20 mV), which is also reduced by dehydrogenases. The position of this new cytochrome in the aerobic respiratory chain is under investigation. Two terminal oxidase complexes branch at the site of ubiquinone-8, and the Km value for oxygen of the purified cytochrome b-558-d complex is about 8-fold lower than that of the purified cytochrome b-562-o complex when ubiquinol-1 is used as substrate. This result is consistent with the idea that the cytochrome b-558-d complex is synthesized as an alternative oxidase for more efficient utilization of oxygen at low oxygen concentration. Thus, E. coli cells can maintain efficient oxidative energy conservation over a wide range of oxygen pressures by simply changing the contents of the two terminal oxidases, each of which functions as a coupling site.

[Effects of nitrogen additions on soil hydrolase and oxidase activities in Pinus elliottii plantations.

PubMed

Zhang, Chuang; Zou, Hong Tao; Zhang, Xin Yu; Kou, Liang; Yang, Yang; Sun, Xiao Min; Li, Sheng Gong; Wang, Hui Min

2016-11-18

We evaluated responses of hydrolase and oxidase activities in a subtropical Pinus elliottii plantation through a nitrogen (N) addition field experiment (dosage level: 0, 40, 120 kg N·hm -2 ·a -1 ). The results showed that N additions significantly decreased the carbon, nitrogen and phosphorus related hydrolase and oxidase activities. The activities of β-1,4-glucosidase (BG), cellobiohydrolase (CBH), β-1,4-N-acetylglucosaminidase (NAG) and peroxidase (PER) activities were decreased by 16.5%-51.1% due to N additions, and the decrease was more remarkable in the higher N addition treatment. The activities of α-1,4-glucosidase (aG), β-1,4-xylosidase (BX), acid phosphatase (AP) and phenol oxidase (PPO) were decreased by 14.5%-38.6% by N additions, however, there was no significant difference among the different N addition treatments. Soil enzyme activities varied obviously in different seasons. The activities of BG, NAG, BX, CBH, AP and PPO were in the order of March > June > October, and aG and PER activities were in the order of October > March > June. Most of the soil hydrolase and oxidase activities were positively correlated with soil pH, but negatively with NO 3 - -N content. It indicated that N additions inhibited soil hydrolase and oxidase activities by reducing soil pH and increasing soil nitrification. N additions inhibited the soil organic matter mineralization and turnover in the subtropical area, and the effects were obvious with the increasing dosage of N additions.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

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.

Extracellular cholesterol oxidase production by Streptomyces aegyptia, in vitro anticancer activities against rhabdomyosarcoma, breast cancer cell-lines and in vivo apoptosis.

PubMed

El-Naggar, Noura El-Ahmady; Soliman, Hoda M; El-Shweihy, Nancy M

2018-02-09

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett-Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL -1 ) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL -1 ). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide

PubMed Central

Korshunov, Sergey; Imlay, Karin R. C.; Imlay, James A.

2016-01-01

Summary When sulfur compounds are scarce or difficult to process, E. coli adapts by inducing the high-level expression of sulfur-compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low-micromolar levels of sulfide inhibited the proton-pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back-up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic-anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats. PMID:26991114

A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

PubMed Central

Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

2000-01-01

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis*

PubMed Central

Dellero, Younès; Mauve, Caroline; Boex-Fontvieille, Edouard; Flesch, Valérie; Jossier, Mathieu; Tcherkez, Guillaume; Hodges, Michael

2015-01-01

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The 12C/13C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the 13C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes. PMID:25416784

Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer's disease.

PubMed

Knez, Damijan; Sova, Matej; Košak, Urban; Gobec, Stanislav

2017-05-01

Accumulating evidence indicates a solid relationship between several enzymes and Alzheimer's disease. Cholinesterases and monoamine oxidases are closely associated with the disease symptomatology and progression and have been tackled simultaneously using several multifunctional ligands. This design strategy offers great chances to alter the course of Alzheimer's disease, in addition to alleviation of the symptoms. More than 15 years of research has led to the identification of various dual cholinesterase/monoamine oxidase inhibitors, while some showing positive outcomes in clinical trials, thus giving rise to additional research efforts in the field. The aim of this review is to provide an update on the novel dual inhibitors identified recently and to shed light on their therapeutic potential.

Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus.

PubMed

Kim, Man Suk; Kim, Young Jae

2004-11-30

Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).

Novel L-amino acid oxidase with algicidal activity against toxic cyanobacterium Microcystis aeruginosa synthesized by a bacterium Aquimarina sp.

PubMed

Chen, Wen Ming; Sheu, Fu Sian; Sheu, Shih Yi

2011-09-10

A brownish yellow pigmented bacterial strain, designated antisso-27, was recently isolated from a water area of saltpan in Southern Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain antisso-27 belongs the genus Aquimarina in the family Flavobacteriacea and its only closest neighbor is Aquimarina spongiae (96.6%). Based on screening for algicidal activity, strain antisso-27 exhibits potent activity against the toxic cyanobacterium Microcystis aeruginosa. Both the strain antisso-27 bacterial culture and its culture filtrate show algicidal activity against the toxic cyanobacterium, indicating that an algicidal substance is released from strain antisso-27. The algicidal activity of strain antisso-27 occurs during the late stationary phase of bacterial growth. Strain antisso-27 can synthesize an algicidal protein with a molecular mass of 190 kDa, and its isoelectric point is approximately 9.4. This study explores the nature of this algicidal protein such as L-amino acid oxidase with broad substrate specificity. The enzyme is most active with L-leucine, L-isoleucine, L-methionine and L-valine and the hydrogen peroxide generated by its catalysis mediates algicidal activity. This is the first report on an Aquimarina strain algicidal to the toxic M. aeruginosa and the algicidal activity is generated through its enzymatic activity of L-amino acid oxidase. Copyright © 2011 Elsevier Inc. All rights reserved.

Mapping the primary structure of copper/topaquinone-containing methylamine oxidase from Aspergillus niger.

PubMed

Lenobel, R; Sebela, M; Frébort, I

2005-01-01

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).

Monoamine oxidase inhibitors from Gentiana lutea.

PubMed

Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

2004-08-01

Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

PubMed

Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

2003-10-01

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

PubMed

Hiraka, Kentaro; Kojima, Katsuhiro; Lin, Chi-En; Tsugawa, Wakako; Asano, Ryutaro; La Belle, Jeffrey T; Sode, Koji

2018-04-30

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

Cotton Ascorbate Oxidase Promotes Cell Growth in Cultured Tobacco Bright Yellow-2 Cells through Generation of Apoplast Oxidation

PubMed Central

Li, Rong; Xin, Shan; Tao, Chengcheng; Jin, Xiang; Li, Hongbin

2017-01-01

Ascorbate oxidase (AO) plays an important role in cell growth through the modulation of reduction/oxidation (redox) control of the apoplast. Here, a cotton (Gossypium hirsutum) apoplastic ascorbate oxidase gene (GhAO1) was obtained from fast elongating fiber tissues. GhAO1 belongs to the multicopper oxidase (MCO) family and includes a signal peptide and several transmembrane regions. Analyses of quantitative real-time polymerase chain reaction (QRT-PCR) and enzyme activity showed that GhAO1 was expressed abundantly in 15-day post-anthesis (dpa) wild-type (WT) fibers in comparison with fuzzless-lintless (fl) mutant ovules. Subcellular distribution analysis in onion cells demonstrated that GhAO1 is localized in the cell wall. In transgenic tobacco bright yellow-2 (BY-2) cells with ectopic overexpression of GhAO1, the enhancement of cell growth with 1.52-fold increase in length versus controls was indicated, as well as the enrichment of both total ascorbate in whole-cells and dehydroascorbate acid (DHA) in apoplasts. In addition, promoted activities of AO and monodehydroascorbate reductase (MDAR) in apoplasts and dehydroascorbate reductase (DHAR) in whole-cells were displayed in transgenic tobacco BY-2 cells. Accumulation of H2O2, and influenced expressions of Ca2+ channel genes with the activation of NtMPK9 and NtCPK5 and the suppression of NtTPC1B were also demonstrated in transgenic tobacco BY-2 cells. Finally, significant induced expression of the tobacco NtAO gene in WT BY-2 cells under indole-3-acetic acid (IAA) treatment appeared; however, the sensitivity of the NtAO gene expression to IAA disappeared in transgenic BY-2 cells, revealing that the regulated expression of the AO gene is under the control of IAA. Taken together, these results provide evidence that GhAO1 plays an important role in fiber cell elongation and may promote cell growth by generating the oxidation of apoplasts, via the auxin-mediated signaling pathway. PMID:28644407

Implications of terminal oxidase function in regulation of salicylic acid on soybean seedling photosynthetic performance under water stress.

PubMed

Tang, Yanping; Sun, Xin; Wen, Tao; Liu, Mingjie; Yang, Mingyan; Chen, Xuefei

2017-03-01

The aim of this study is to investigate whether exogenous application of salicylic acid (SA) could modulate the photosynthetic capacity of soybean seedlings in water stress tolerance, and to clarify the potential functions of terminal oxidase (plastid terminal oxidase (PTOX) and alternative oxidase (AOX)) in SA' s regulation on photosynthesis. The effects of SA and water stress on gas exchange, pigment contents, chlorophyll fluorescence, enzymes (guaiacol peroxidase (POD; EC 1.11.1.7), superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and NADP-malate dehydrogenase (NADP-MDH; EC1.1.1.82)) activity and transcript levels of PTOX, AOX1, AOX2a, AOX2b were examined in a hydroponic cultivation system. Results indicate that water stress significantly decreased the photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), pigment contents (Chla + b, Chla/b, Car), maximum quantum yield of PSⅡphotochemistry (Fv/Fm), efficiency of excitation capture of open PSⅡcenter (Fv'/Fm'), quantum efficiency of PSⅡphotochemistry (ΦPSⅡ), photochemical quenching (qP), and increased malondialdehyde (MDA) content and the activity of all the enzymes. SA pretreatment led to significant decreases in Ci and MDA content, and increases in Pn, Gs, E, pigment contents, Fv/Fm, Fv'/Fm', ΦPSⅡ, qP, and the activity of all the enzymes. SA treatment and water stress alone significantly up-regulated the expression of PTOX, AOX1 and AOX2b. SA pretreatment further increased the transcript levels of PTOX and AOX2b of soybean seedling under water stress. These results indicate that SA application alleviates the water stress-induced decrease in photosynthesis may mainly through maintaining a lower reactive oxygen species (ROS) level, a greater PSⅡefficiency, and an enhanced alternative respiration and chlororespiration. PTOX and AOX may play important roles in SA-mediated resistance to water stress. Copyright © 2016

The dietary polysaccharide maltodextrin promotes Salmonella survival and mucosal colonization in mice.

PubMed

Nickerson, Kourtney P; Homer, Craig R; Kessler, Sean P; Dixon, Laura J; Kabi, Amrita; Gordon, Ilyssa O; Johnson, Erin E; de la Motte, Carol A; McDonald, Christine

2014-01-01

In the latter half of the 20th century, societal and technological changes led to a shift in the composition of the American diet to include a greater proportion of processed, pre-packaged foods high in fat and carbohydrates, and low in dietary fiber (a "Western diet"). Over the same time period, there have been parallel increases in Salmonella gastroenteritis cases and a broad range of chronic inflammatory diseases associated with intestinal dysbiosis. Several polysaccharide food additives are linked to bacterially-driven intestinal inflammation and may contribute to the pathogenic effects of a Western diet. Therefore, we examined the effect of a ubiquitous polysaccharide food additive, maltodextrin (MDX), on clearance of the enteric pathogen Salmonella using both in vitro and in vivo infection models. When examined in vitro, murine bone marrow-derived macrophages exposed to MDX had altered vesicular trafficking, suppressed NAPDH oxidase expression, and reduced recruitment of NADPH oxidase to Salmonella-containing vesicles, which resulted in persistence of Salmonella in enlarged Rab7+ late endosomal vesicles. In vivo, mice consuming MDX-supplemented water had a breakdown of the anti-microbial mucous layer separating gut bacteria from the intestinal epithelium surface. Additionally, oral infection of these mice with Salmonella resulted in increased cecal bacterial loads and enrichment of lamina propria cells harboring large Rab7+ vesicles. These findings indicate that consumption of processed foods containing the polysaccharide MDX contributes to suppression of intestinal anti-microbial defense mechanisms and may be an environmental priming factor for the development of chronic inflammatory disease.

The Dietary Polysaccharide Maltodextrin Promotes Salmonella Survival and Mucosal Colonization in Mice

PubMed Central

Nickerson, Kourtney P.; Homer, Craig R.; Kessler, Sean P.; Dixon, Laura J.; Kabi, Amrita; Gordon, Ilyssa O.; Johnson, Erin E.; de la Motte, Carol A.; McDonald, Christine

2014-01-01

In the latter half of the 20th century, societal and technological changes led to a shift in the composition of the American diet to include a greater proportion of processed, pre-packaged foods high in fat and carbohydrates, and low in dietary fiber (a “Western diet”). Over the same time period, there have been parallel increases in Salmonella gastroenteritis cases and a broad range of chronic inflammatory diseases associated with intestinal dysbiosis. Several polysaccharide food additives are linked to bacterially-driven intestinal inflammation and may contribute to the pathogenic effects of a Western diet. Therefore, we examined the effect of a ubiquitous polysaccharide food additive, maltodextrin (MDX), on clearance of the enteric pathogen Salmonella using both in vitro and in vivo infection models. When examined in vitro, murine bone marrow-derived macrophages exposed to MDX had altered vesicular trafficking, suppressed NAPDH oxidase expression, and reduced recruitment of NADPH oxidase to Salmonella-containing vesicles, which resulted in persistence of Salmonella in enlarged Rab7+ late endosomal vesicles. In vivo, mice consuming MDX-supplemented water had a breakdown of the anti-microbial mucous layer separating gut bacteria from the intestinal epithelium surface. Additionally, oral infection of these mice with Salmonella resulted in increased cecal bacterial loads and enrichment of lamina propria cells harboring large Rab7+ vesicles. These findings indicate that consumption of processed foods containing the polysaccharide MDX contributes to suppression of intestinal anti-microbial defense mechanisms and may be an environmental priming factor for the development of chronic inflammatory disease. PMID:25000398

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

PubMed Central

Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

2014-01-01

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

Molecular evolution of the polyamine oxidase gene family in Metazoa

PubMed Central

2012-01-01

Background Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. Results We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including

Molecular evolution of the polyamine oxidase gene family in Metazoa.

PubMed

Polticelli, Fabio; Salvi, Daniele; Mariottini, Paolo; Amendola, Roberto; Cervelli, Manuela

2012-06-20

Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. We analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all

Ectopic Expression of Pumpkin Gibberellin Oxidases Alters Gibberellin Biosynthesis and Development of Transgenic Arabidopsis Plants1

PubMed Central

Radi, Abeer; Lange, Theo; Niki, Tomoya; Koshioka, Masaji; Lange, Maria João Pimenta

2006-01-01

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development. PMID:16384902

Synthetic Heme/Copper Assemblies: Toward an Understanding of Cytochrome c Oxidase Interactions with Dioxygen and Nitrogen Oxides

PubMed Central

Hematian, Shabnam; Garcia-Bosch, Isaac; Karlin, Kenneth D.

2016-01-01

Conspectus Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper and/or iron ions, those reacting with dioxygen (O2) and/or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2−)). As inspiration for this work, we turn to mitochondrial cytochrome c oxidase which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis and characterization of new O2-adducts whose further study will add insights into O2-reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO’s function, which is intimately tied to cellular O2-balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-FeIII-O-CuII(L) products; their properties are discussed. The O-atom is derived from dioxygen and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo-complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a “naked” synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active-sites. The other sector of research is focused on heme/Cu assemblies mediating the

A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

PubMed

Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

2010-03-26

Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.

Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

PubMed Central

Vanlerberghe, Greg C.; McIntosh, Lee

1992-01-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

Regional up-regulation of NOX2 contributes to the differential vulnerability of outer hair cells to neomycin.

PubMed

Qi, Meihao; Qiu, Yang; Zhou, Xueying; Tian, Keyong; Zhou, Ke; Sun, Fei; Yue, Bo; Chen, Fuquan; Zha, Dingjun; Qiu, Jianhua

2018-06-02

In hearing loss induced by aminoglycoside antibiotics, the outer hair cells (OHCs) in the basal turn are always more susceptible than OHCs in the apical turn, while the underlying mechanisms remain unknown. In this study, we reported that NAPDH oxidase 2 (NOX2) played an important role in the OHCs damage preferentially in the basal turn. Normally, NOX2 was evenly expressed in OHCs among different turns, at a relatively low level. However, after neomycin treatment, NOX2 was dominantly induced in OHCs in the basal turn. In vivo and in vitro studies demonstrated that inhibition of NOX2 significantly alleviated neomycin-induced OHCs damages, as seen from both the cleaved caspase-3 and TUNEL staining. Moreover, gp91 ds-tat delivery and DHE staining results showed that NOX2-derived ROS was responsible for neomycin ototoxicity. Taken together, our study shows that regional up-expression of NOX2 and subsequent increase of ROS in OHCs of the basal turn is an important factor contributing to the vulnerability of OHCs there, which should shed light on the prevention of hearing loss induced by aminoglycoside antibiotics. Copyright © 2018 Elsevier Inc. All rights reserved.

Timing of electron and proton transfer in the ba(3) cytochrome c oxidase from Thermus thermophilus.

PubMed

von Ballmoos, Christoph; Lachmann, Peter; Gennis, Robert B; Ädelroth, Pia; Brzezinski, Peter

2012-06-05

Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O(2) to H(2)O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O(2) reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H(+)/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H(+)/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba(3) oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.

Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase.

PubMed

Salvi, Francesca; Rodriguez, Isela; Hamelberg, Donald; Gadda, Giovanni

2016-03-15

Choline oxidase from Arthrobacter globiformis catalyzes the oxidation of choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of choline oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type choline oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of choline oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor.

Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment.

PubMed

Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A; Karuparthi, Poorna R; Hayden, Melvin R; Rehmer, Nathan; DeMarco, Vincent G; Andresen, Bradley T; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R

2008-02-01

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

Influence of Tridax procumbens on lysyl oxidase activity and wound healing.

PubMed

Udupa, S L; Udupa, A L; Kulkarni, D R

1991-08-01

The effects of an indigenous drug, Tridax procumbens L. (Compositae), on developing granulation tissue in rats were studied. Subcutaneously harvested granuloma tissue formed on dead space wound was removed at 4 day intervals up to 32 days of wounding. Lysyl oxidase activity, protein content, specific activity, and breaking strength were all increased in drug-treated animals as compared to controls. A fall in the lysyl oxidase activity was observed in drug-treated animals after day 8. The drug may be having a dual role: one a stimulatory (direct) effect in the initial phase of wound healing and the other a depressant (indirect) effect in the later stage.

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

PubMed

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

2015-12-25

Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation. Copyright © 2015 Elsevier Inc. All rights reserved.

Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

PubMed Central

Okuno, Tatsusada; Takata, Kazushiro; Koda, Toru; Tada, Satoru; Shirakura, Takashi; Fujimura, Harutoshi; Mochizuki, Hideki; Sakoda, Saburo; Nakatsuji, Yuji

2013-01-01

Objectives Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS. Methods XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies. Results We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination. Conclusion These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology. PMID:23951137

Serotonin Receptor 6 Mediates Defective Brain Development in Monoamine Oxidase A-deficient Mouse Embryos

PubMed Central

Wang, Chi Chiu; Man, Gene Chi Wai; Chu, Ching Yan; Borchert, Astrid; Ugun-Klusek, Aslihan; Billett, E. Ellen; Kühn, Hartmut; Ufer, Christoph

2014-01-01

Monoamine oxidases A and B (MAO-A and MAO-B) are enzymes of the outer mitochondrial membrane that metabolize biogenic amines. In the adult central nervous system, MAOs have important functions for neurotransmitter homeostasis. Expression of MAO isoforms has been detected in the developing embryo. However, suppression of MAO-B does not induce developmental alterations. In contrast, targeted inhibition and knockdown of MAO-A expression (E7.5–E10.5) caused structural abnormalities in the brain. Here we explored the molecular mechanisms underlying defective brain development induced by MAO-A knockdown during in vitro embryogenesis. The developmental alterations were paralleled by diminished apoptotic activity in the affected neuronal structures. Moreover, dysfunctional MAO-A expression led to elevated levels of embryonic serotonin (5-hydroxytryptamine (5-HT)), and we found that knockdown of serotonin receptor-6 (5-Htr6) expression or pharmacologic inhibition of 5-Htr6 activity rescued the MAO-A knockdown phenotype and restored apoptotic activity in the developing brain. Our data suggest that excessive 5-Htr6 activation reduces activation of caspase-3 and -9 of the intrinsic apoptotic pathway and enhances expression of antiapoptotic proteins Bcl-2 and Bcl-XL. Moreover, we found that elevated 5-HT levels in MAO-A knockdown embryos coincided with an enhanced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and a reduction of proliferating cell numbers. In summary, our findings suggest that excessive 5-HT in MAO-A-deficient mouse embryos triggers cellular signaling cascades via 5-Htr6, which suppresses developmental apoptosis in the brain and thus induces developmental retardations. PMID:24497636

Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

PubMed Central

Liu, Jian; Zhou, Jun; Xing, Da

2012-01-01

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

A repeatedly refuelable mediated biofuel cell based on a hierarchical porous carbon electrode

NASA Astrophysics Data System (ADS)

Fujita, Shuji; Yamanoi, Shun; Murata, Kenichi; Mita, Hiroki; Samukawa, Tsunetoshi; Nakagawa, Takaaki; Sakai, Hideki; Tokita, Yuichi

2014-05-01

Biofuel cells that generate electricity from renewable fuels, such as carbohydrates, must be reusable through repeated refuelling, should these devices be used in consumer electronics. We demonstrate the stable generation of electricity from a glucose-powered mediated biofuel cell through multiple refuelling cycles. This refuelability is achieved by immobilizing nicotinamide adenine dinucleotide (NAD), an electron-transfer mediator, and redox enzymes in high concentrations on porous carbon particles constituting an anode while maintaining their electrochemical and enzymatic activities after the immobilization. This bioanode can be refuelled continuously for more than 60 cycles at 1.5 mA cm-2 without significant potential drop. Cells assembled with these bioanodes and bilirubin-oxidase-based biocathodes can be repeatedly used to power a portable music player at 1 mW cm-3 through 10 refuelling cycles. This study suggests that the refuelability within consumer electronics should facilitate the development of long and repeated use of the mediated biofuel cells as well as of NAD-based biosensors, bioreactors, and clinical applications.

RpoS contributes to phagocyte oxidase-mediated stress resistance during urinary tract infection by Escherichia coli CFT073.

PubMed

Hryckowian, Andrew J; Welch, Rodney A

2013-02-12

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (σ(S)), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study of rpoS in UPEC strain CFT073 began after we discovered an rpoS-frameshift mutation in one of our laboratory stocks of "wild-type" CFT073. We demonstrate that an rpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073 rpoS in urine. This indicates that rpoS is needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations in E. coli K-12, CFT073 rpoS is more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073 rpoS in bladder colonization is lost. These results demonstrate that σ(S) is important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though σ(S) affects the pathogenesis of other bacterial species, this is the first work that directly implicates σ(S) as important for UPEC pathogenesis. UPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (σ(S)), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adapted E. coli K-12. Here, we show that

Exercise training decreases NADPH oxidase activity and restores skeletal muscle mass in heart failure rats.

PubMed

Cunha, Telma F; Bechara, Luiz R G; Bacurau, Aline V N; Jannig, Paulo R; Voltarelli, Vanessa A; Dourado, Paulo M; Vasconcelos, Andrea R; Scavone, Cristóforo; Ferreira, Júlio C B; Brum, Patricia C

2017-04-01

We have recently demonstrated that NADPH oxidase hyperactivity, NF-κB activation, and increased p38 phosphorylation lead to atrophy of glycolytic muscle in heart failure (HF). Aerobic exercise training (AET) is an efficient strategy to counteract skeletal muscle atrophy in this syndrome. Therefore, we tested whether AET would regulate muscle redox balance and protein degradation by decreasing NADPH oxidase hyperactivity and reestablishing NF-κB signaling, p38 phosphorylation, and proteasome activity in plantaris muscle of myocardial infarcted-induced HF (MI) rats. Thirty-two male Wistar rats underwent MI or fictitious surgery (SHAM) and were randomly assigned into untrained (UNT) and trained (T; 8 wk of AET on treadmill) groups. AET prevented HF signals and skeletal muscle atrophy in MI-T, which showed an improved exercise tolerance, attenuated cardiac dysfunction and increased plantaris fiber cross-sectional area. To verify the role of inflammation and redox imbalance in triggering protein degradation, circulating TNF-α levels, NADPH oxidase profile, NF-κB signaling, p38 protein levels, and proteasome activity were assessed. MI-T showed a reduced TNF-α levels, NADPH oxidase activity, and Nox2 mRNA expression toward SHAM-UNT levels. The rescue of NADPH oxidase activity induced by AET in MI rats was paralleled by reducing nuclear binding activity of the NF-κB, p38 phosphorylation, atrogin-1, mRNA levels, and 26S chymotrypsin-like proteasome activity. Taken together our data provide evidence for AET improving plantaris redox homeostasis in HF associated with a decreased NADPH oxidase, redox-sensitive proteins activation, and proteasome hyperactivity further preventing atrophy. These data reinforce the role of AET as an efficient therapy for muscle wasting in HF. NEW & NOTEWORTHY This study demonstrates, for the first time, the contribution of aerobic exercise training (AET) in decreasing muscle NADPH oxidase activity associated with reduced reactive oxygen

Cell-free NADPH oxidase activation assays: "in vitro veritas".

PubMed

Pick, Edgar

2014-01-01

The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC

PubMed Central

Li, Zhu; Shanmuganathan, Aranganathan; Ruetz, Markus; Yamada, Kazuhiro; Lesniak, Nicholas A.; Kräutler, Bernhard; Brunold, Thomas C.; Koutmos, Markos; Banerjee, Ruma

2017-01-01

The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2. The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and β-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC. PMID:28442570

Single mutations that redirect internal proton transfer in the ba3 oxidase from Thermus thermophilus

PubMed Central

Smirnova, Irina; Chang, Hsin-Yang; von Ballmoos, Christoph; Ädelroth, Pia; Gennis, Robert B.; Brzezinski, Peter

2014-01-01

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa3-type oxidases proton uptake to the catalytic site and “pump site” occur simultaneously. However, with the ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than with the aa3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though the ba3 CytcO uses only a single proton pathway for transfer of the substrate and “pumped” protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site, respectively. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site. PMID:24004023

Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign.

PubMed

Tremey, Emilie; Suraniti, Emmanuel; Courjean, Olivier; Gounel, Sébastien; Stines-Chaumeil, Claire; Louerat, Frédéric; Mano, Nicolas

2014-06-04

In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

Involvement of alternative oxidase in the regulation of sensitivity of Sclerotinia sclerotiorum to the fungicides azoxystrobin and procymidone.

PubMed

Xu, Ting; Wang, Ya-Ting; Liang, Wu-Sheng; Yao, Fei; Li, Yong-Hong; Li, Dian-Rong; Wang, Hao; Wang, Zheng-Yi

2013-06-01

Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.

Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

PubMed

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.

Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B

PubMed Central

Hagenow, S.; Stasiak, A.; Ramsay, R. R.; Stark, H.

2017-01-01

Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands. PMID:28084411

In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria

PubMed Central

Dahiya, Tulika; Pundir, C.S.

2013-01-01

Background & objectives: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. Methods: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. Results: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B6 deficient fed rats as compared with 30 per cent casein vitamin B6 deficient fed rats and control rats. Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones. PMID:23481063

Immobilization techniques to avoid enzyme loss from oxidase-based biosensors: a one-year study.

PubMed

House, Jody L; Anderson, Ellen M; Ward, W Kenneth

2007-01-01

Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased.

A New Transgenic Mouse Model for Studying the Neurotoxicity of Spermine Oxidase Dosage in the Response to Excitotoxic Injury

PubMed Central

Cervelli, Manuela; Bellavia, Gabriella; D'Amelio, Marcello; Cavallucci, Virve; Moreno, Sandra; Berger, Joachim; Nardacci, Roberta; Marcoli, Manuela; Maura, Guido; Piacentini, Mauro; Amendola, Roberto; Cecconi, Francesco; Mariottini, Paolo

2013-01-01

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain. PMID

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Screening, separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi-mode microplate reader.

PubMed

Wang, Jing; Shi, Dongfang; Zheng, Meizhu; Ma, Bing; Cui, Jing; Liu, Chunming; Liu, Chengyu

2017-11-01

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora), and both high-performance counter-current chromatography and medium-pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi-mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high-performance counter-current chromatography and medium-pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1-O-β-ᴅ-glucopyranosyl-8-O-benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

PubMed

Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

2010-08-20

Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

PubMed Central

Dailey, T. A.; Dailey, H. A.

1996-01-01

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase. PMID:8771201

A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

PubMed

Bifulco, Davide; Pollegioni, Loredano; Tessaro, Davide; Servi, Stefano; Molla, Gianluca

2013-08-01

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent Indicator for Hydrogen Peroxide and Oxidase-Based Biosensing.

PubMed

Mao, Zhengui; Qing, Zhihe; Qing, Taiping; Xu, Fengzhou; Wen, Li; He, Xiaoxiao; He, Dinggeng; Shi, Hui; Wang, Kemin

2015-07-21

Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probe's oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity

PubMed Central

Hess, Kenneth C.; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A.; Buck, Jochen; Levin, Lonny R.; Barrientos, Antoni

2014-01-01

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.—Hess, K. C., Liu, J., Manfredi, G., Mühlschlegel, F. A., Buck, J., Levin, L. R., Barrientos, A. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. PMID:25002117

Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

PubMed Central

Daou, Marianne; Piumi, François; Cullen, Daniel; Record, Eric

2016-01-01

ABSTRACT The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these

Ferrocene-Modified Linear Poly(ethylenimine) for Enzymatic Immobilization and Electron Mediation.

PubMed

Hickey, David P

2017-01-01

Enzymatic glucose biosensors and biofuel cells make use of the electrochemical transduction between an oxidoreductase enzyme, such as glucose oxidase (GOx), and an electrode to either quantify the amount of glucose in a solution or generate electrical energy. However, many enzymes including GOx are not able to electrochemically interact with an electrode surface directly, but require an external electrochemical relay to shuttle electrons to the electrode. Ferrocene-modified linear poly(ethylenimine) (Fc-LPEI) redox polymers have been designed to simultaneously immobilize glucose oxidase (GOx) at an electrode and mediate electron transfer from their flavin adenine dinucleotide (FAD) active site to the electrode surface. Cross-linked films of Fc-LPEI create hydrogel networks that allow for rapid transport of glucose, while the covalently bound ferrocene moieties are able to facilitate rapid electron transfer due to the ability of ferrocene to exchange electrons between adjacent ferrocene residues. For these reasons, Fc-LPEI films have been widely used in the development of high current density bioanode materials. This chapter describes the synthesis of a commonly used dimethylferrocene-modified linear poly(ethylenimine), as well as the subsequent preparation and electrochemical characterization of a GOx bioanode film utilizing the synthesized polymer.

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages*

PubMed Central

Ejlerskov, Patrick; Christensen, Dan Ploug; Beyaie, David; Burritt, James B.; Paclet, Marie-Helene; Gorlach, Agnes; van Deurs, Bo; Vilhardt, Frederik

2012-01-01

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b558) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91phox and CeCl3 cytochemistry showed the presence of gp91phox and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b558 is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b558-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5′-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b558 under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b558 exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b558, which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b558 did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b558 depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell. PMID:22157766

Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics.

PubMed

Suzuki, Ryuichiro; Hasuike, Yuka; Hirabayashi, Moeka; Fukuda, Tatsuo; Okada, Yoshihito; Shirataki, Yoshiaki

2013-10-01

We demonstrate that NMR-based metabolomics studies can be used to identify xanthine oxidase-inhibitory compounds in the diethyl ether soluble fraction prepared from a methanolic extract of Sophora flavescens. Loading plot analysis, accompanied by direct comparison of 1H NMR spectraexhibiting characteristic signals, identified compounds exhibiting inhibitory activity. NMR analysis indicated that these characteristic signals were attributed to flavanones such as sophoraflavanone G and kurarinone. Sophoraflavanone G showed inhibitory activity towards xanthine oxidase in an in vitro assay.

Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1

PubMed Central

Zhou, Fasong; Zhang, Ziguo; Gregersen, Per L.; Mikkelsen, Jørn D.; de Neergaard, Eigil; Collinge, David B.; Thordal-Christensen, Hans

1998-01-01

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response. PMID:9576772

Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

PubMed Central

Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

2001-01-01

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

PubMed Central

Lange, T; Hedden, P; Graebe, J E

1994-01-01

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases. Images PMID:8078921

Pseudo-bi-enzyme glucose sensor: ZnS hollow spheres and glucose oxidase concerted catalysis glucose.

PubMed

Shuai, Ying; Liu, Changhua; Wang, Jia; Cui, Xiaoyan; Nie, Ling

2013-06-07

This work creatively uses peroxidase-like ZnS hollow spheres (ZnS HSs) to cooperate with glucose oxidase (GOx) for glucose determinations. This approach is that the ZnS HSs electrocatalytically oxidate the enzymatically generated H2O2 to O2, and then the O2 circularly participates in the previous glucose oxidation by glucose oxidase. Au nanoparticles (AuNPs) and carbon nanotubes (CNTs) are used as electron transfer and enzyme immobilization matrices, respectively. The biosensor of glucose oxidase-carbon nanotubes-Au nanoparticles-ZnS hollow spheres-gold electrode (GOx-CNT-AuNPs-ZnS HSs-GE) exhibits a rapid response, a low detection limit (10 μM), a wide linear range (20 μM to 7 mM) as well as good anti-interference, long-term longevity and reproducibility.

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, K.; Pilot, T.F.; Meany, J.E.

1990-01-01

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing inmore » solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.« less

4-Hydroxyanisole: the most suitable monophenolic substrate for determining spectrophotometrically the monophenolase activity of polyphenol oxidase from fruits and vegetables.

PubMed

Espín, J C; Tudela, J; García-Cánovas, F

1998-05-15

A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those of p-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolas activity of polyphenol oxidase from fruits and vegetables.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

PubMed

Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

2017-08-01

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

A Peroxidase-linked Spectrophotometric Assay for the Detection of Monoamine Oxidase Inhibitors.

PubMed

Zhi, Kangkang; Yang, Zhongduo; Sheng, Jie; Shu, Zongmei; Shi, Yin

2016-01-01

To develop a new more accurate spectrophotometric method for detecting monoamine oxidase inhibitors from plant extracts, a series of amine substrates were selected and their ability to be oxidized by monoamine oxidase was evaluated by the HPLC method and a new substrate was used to develop a peroxidase-linked spectrophotometric assay. 4-(Trifluoromethyl) benzylamine (11) was proved to be an excellent substrate for peroxidase-linked spectrophotometric assay. Therefore, a new peroxidase-linked spectrophotometric assay was set up. The principle of the method is that the MAO converts 11 into aldehyde, ammonia and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide will oxidize 4-aminoantipyrine into oxidised 4-aminoantipyrine which can condense with vanillic acid to give a red quinoneimine dye. The production of the quinoneimine dye was detected at 490 nm by a microplate reader. The ⊿OD value between the blank group and blank negative control group in this new method is twice as much as that in Holt's method, which enables the procedure to be more accurate and avoids the produce of false positive results. The new method will be helpful for researchers to screening monoamine oxidase inhibitors from deep-color plant extracts.

Spermine oxidase, a polyamine catabolic enzyme that links Helicobacter pylori CagA and gastric cancer risk

PubMed Central

Chaturvedi, Rupesh; de Sablet, Thibaut; Peek, Richard M.; Wilson, Keith T.

2012-01-01

We have recently reported that Helicobacter pylori strains expressing the virulence factor cytotoxin-associated gene A (CagA) stimulate increased levels of spermine oxidase (SMO) in gastric epithelial cells, while cagA– strains did not. SMO catabolizes the polyamine spermine and produces H2O2 that results in both apoptosis and DNA damage. Exogenous overexpression of CagA confirmed these findings, and knockdown or inhibition of SMO blocked CagA-mediated apoptosis and DNA damage. The strong association of SMO, apoptosis, and DNA damage was also demonstrated in humans infected with cagA+, but not cagA– strains. In infected gerbils and mice, DNA damage was CagA-dependent and only present in epithelial cells that expressed SMO. We also discovered SMOhigh gastric epithelial cells from infected animals with dysplasia that are resistant to apoptosis despite high levels of DNA damage. Inhibition of polyamine synthesis or SMO could abrogate the development of this cell population that may represent precursors for neoplastic transformation. PMID:22555547

Immobilization of Glucose Oxidase on a Carbon Nanotubes/Dendrimer-Ferrocene Modified Electrode for Reagentless Glucose Biosensing.

PubMed

Zhou, Juan; Li, Huan; Yang, Huasong; Cheng, Hui; Lai, Guosong

2017-01-01

Ferrocene-grafted dendrimer was covalently linked to the surface of a carbon nanotubes (CNTs)-chitosan (CS) nanocomposite modified electrode for immobilizing high-content glucose oxidase (GOx), which resulted in the successful development a novel reagentless glucose biosensor. Electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry were used to characterize the preparation process and the enzymatically catalytic response of this biosensor. Due to the excellent electron transfer acceleration of the CNTs and the high-content loading of the GOx biomolecule and ferrocene mediator on the electrode matrix, this biosensor showed excellent analytical performance such as fast response time less than 10 s, wide linear range from 0.02 to 2.91 mM and low detection limit down to 7.5 μM as well as satisfactory stability and reproducibility toward the amperometric glucose determination. In addition, satisfactory result was obtained when it was used for the glucose measurements in human blood samples. Thus this biosensor provides great potentials for practical applications.

NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

PubMed Central

Wen, Zhenke; Shimojima, Yasuhiro; Shirai, Tsuyoshi; Li, Yinyin; Ju, Jihang; Yang, Zhen; Tian, Lu; Goronzy, Jörg J.

2016-01-01

Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis. PMID:27088800

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

PubMed

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.

Immobilization Techniques to Avoid Enzyme Loss from Oxidase-Based Biosensors: A One-Year Study

PubMed Central

House, Jody L.; Anderson, Ellen M.; Ward, W. Kenneth

2007-01-01

Background Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. Methods A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. Results The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. Conclusions After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased. PMID:19888375

Identification of a Catalase-Phenol Oxidase in Betalain Biosynthesis in Red Amaranth (Amaranthus cruentus)

PubMed Central

Teng, Xiao-Lu; Chen, Ning; Xiao, Xing-Guo

2016-01-01

Betalains are a group of nitrogen-containing pigments that color plants in most families of Caryophyllales. Their biosynthesis has long been proposed to begin with hydroxylation of L-tyrosine to L-DOPA through monophenolase activity of tyrosinase, but biochemical evidence in vivo remains lacking. Here we report that a Group 4 catalase, catalase-phenol oxidase (named as AcCATPO), was identified, purified and characterized from leaves of Amaranthus cruentus, a betalain plant. The purified enzyme appeared to be a homotrimeric protein composed of subunits of about 58 kDa, and demonstrated not only the catalase activity toward H2O2, but also the monophenolase activity toward L-tyrosine and diphenolase activity toward L-DOPA. Its catalase and phenol oxidase activities were inhibited by common classic catalase and tyrosinase inhibitors, respectively. All its peptide fragments identified by nano-LC-MS/MS were targeted to catalases, and matched with a cDNA-encoded polypeptide which contains both classic catalase and phenol oxidase active sites. These sites were also present in catalases of non-betalain plants analyzed. AcCATPO transcript abundance was positively correlated with the ratio of betaxanthin to betacyanin in both green and red leaf sectors of A. tricolor. These data shows that the fourth group catalase, catalase-phenol oxidase, is present in plant, and might be involved in betaxanthin biosynthesis. PMID:26779247

Role of Angiotensin II type 1 receptor on renal NAD(P)H oxidase, oxidative stress and inflammation in nitric oxide inhibition induced-hypertension.

PubMed

Rincón, J; Correia, D; Arcaya, J L; Finol, E; Fernández, A; Pérez, M; Yaguas, K; Talavera, E; Chávez, M; Summer, R; Romero, F

2015-03-01

Activation of the renin-angiotensin system (RAS), renal oxidative stress and inflammation are constantly present in experimental hypertension. Nitric oxide (NO) inhibition with N(w)-nitro-L-arginine methyl ester (L-NAME) has previously been reported to produce hypertension, increased expression of Angiotensin II (Ang II) and renal dysfunction. The use of Losartan, an Ang II type 1 receptor (AT1R) antagonist has proven to be effective reducing hypertension and renal damage; however, the mechanism by which AT1R blockade reduced kidney injury and normalizes blood pressure in this experimental model is still complete unknown. The current study was designed to test the hypothesis that AT1R activation promotes renal NAD(P)H oxidase up-regulation, oxidative stress and cytokine production during L-NAME induced-hypertension. Male Sprague-Dawley rats were distributed in three groups: L-NAME, receiving 70 mg/100ml of L-NAME, L-NAME+Los, receiving 70 mg/100ml of L-NAME and 40 mg/kg/day of Losartan; and Controls, receiving water instead of L-NAME or L-NAME and Losartan. After two weeks, L-NAME induced high blood pressure, renal overexpression of AT1R, NAD(P)H oxidase sub-units gp91, p22 and p47, increased levels of oxidative stress, interleukin-6 (IL-6) and interleukin-17 (IL-17). Also, we found increased renal accumulation of lymphocytes and macrophages. Losartan treatment abolished the renal expression of gp91, p22, p47, oxidative stress and reduced NF-κB activation and IL-6 expression. These findings indicate that NO induced-hypertension is associated with up-regulation of NADPH oxidase, oxidative stress production and overexpression of key inflammatory mediators. These events are associated with up-regulation of AT1R, as evidenced by their reversal with AT1R blocker treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

NADPH oxidase: an enzyme for multicellularity?

PubMed

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.

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Koh; E-mail: idakoh@sci.kitasato-u.ac.jp; Moriguchi, Tomotaka

2005-07-29

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The {alpha} subunit is composed of two domains, contains NAD{sup +}, and binds folinic acid. The {beta} subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10 A apart. The {gamma} subunit is in contact with two domains of {alpha} subunit and has possibly a folate-binding structure. The {delta} subunit contains a single atom of zinc and has a Cys{sub 3}His zinc finger structure. Based on the structures determined and on themore » previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.« less

Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

NASA Astrophysics Data System (ADS)

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.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase.

PubMed

Amani, Mojtaba; Barzegar, Aboozar; Mazani, Mohammad

2017-04-01

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T * , temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.

NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

PubMed Central

2012-01-01

Background Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Moreover, bradykinin (BK) induces the expression of several inflammatory proteins in brain astrocytes. Recent studies have suggested that increased oxidative stress is implicated in the brain inflammation and injury. However, whether BK induced MMP-9 expression mediated through oxidative stress remains virtually unknown. Herein we investigated the role of redox signals in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells). Results In the study, we first demonstrated that reactive oxygen species (ROS) plays a crucial role in BK-induced MMP-9 expression in cultured brain astrocytes (in vitro) and animal brain tissue (in vivo) models. Next, BK-induced MMP-9 expression is mediated through a Ca2+-mediated PKC-α linking to p47phox/NADPH oxidase 2 (Nox2)/ROS signaling pathway. Nox2-dependent ROS generation led to activation and up-regulation of the downstream transcriptional factor AP-1 (i.e. c-Fos and c-Jun), which bound to MMP-9 promoter region, and thereby turned on transcription of MMP-9 gene. Functionally, BK-induced MMP-9 expression enhanced astrocytic migration. Conclusions These results demonstrated that in RBA-1 cells, activation of AP-1 (c-Fos/c-Jun) by the PKC-α-mediated Nox2/ROS signals is essential for up-regulation of MMP-9 and cell migration enhanced by BK. PMID:23176293

Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

NASA Astrophysics Data System (ADS)

Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

2013-11-01

Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

PubMed

López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.