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

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

2016-05-01

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

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

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

2010-03-23

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

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

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

2017-12-01

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

Quantitative flux analysis reveals folate-dependent NADPH production

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

2014-06-01

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

A role for NADPH oxidase in antigen presentation

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Gail J Gardiner

2013-09-01

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

Oxidized CaMKII causes cardiac sinus node dysfunction in mice

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Swaminathan, Paari Dominic; Purohit, Anil; Soni, Siddarth; Voigt, Niels; Singh, Madhu V.; Glukhov, Alexey V.; Gao, Zhan; He, B. Julie; Luczak, Elizabeth D.; Joiner, Mei-ling A.; Kutschke, William; Yang, Jinying; Donahue, J. Kevin; Weiss, Robert M.; Grumbach, Isabella M.; Ogawa, Masahiro; Chen, Peng-Sheng; Efimov, Igor; Dobrev, Dobromir; Mohler, Peter J.; Hund, Thomas J.; Anderson, Mark E.

2011-01-01

Sinus node dysfunction (SND) is a major public health problem that is associated with sudden cardiac death and requires surgical implantation of artificial pacemakers. However, little is known about the molecular and cellular mechanisms that cause SND. Most SND occurs in the setting of heart failure and hypertension, conditions that are marked by elevated circulating angiotensin II (Ang II) and increased oxidant stress. Here, we show that oxidized calmodulin kinase II (ox-CaMKII) is a biomarker for SND in patients and dogs and a disease determinant in mice. In wild-type mice, Ang II infusion caused sinoatrial nodal (SAN) cell oxidation by activating NADPH oxidase, leading to increased ox-CaMKII, SAN cell apoptosis, and SND. p47–/– mice lacking functional NADPH oxidase and mice with myocardial or SAN-targeted CaMKII inhibition were highly resistant to SAN apoptosis and SND, suggesting that ox-CaMKII–triggered SAN cell death contributed to SND. We developed a computational model of the sinoatrial node that showed that a loss of SAN cells below a critical threshold caused SND by preventing normal impulse formation and propagation. These data provide novel molecular and mechanistic information to understand SND and suggest that targeted CaMKII inhibition may be useful for preventing SND in high-risk patients. PMID:21785215

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

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Isabel Méndez

2018-03-01

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

Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

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Susan J. Gelman

2018-01-01

Full Text Available Summary: Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1 occur in multiple types of human cancer. Here, we show that these mutations significantly disrupt NADPH homeostasis by consuming NADPH for 2-hydroxyglutarate (2-HG synthesis. Cells respond to 2-HG synthesis, but not exogenous administration of 2-HG, by increasing pentose phosphate pathway (PPP flux. We show that 2-HG production competes with reductive biosynthesis and the buffering of oxidative stress, processes that also require NADPH. IDH1 mutants have a decreased capacity to synthesize palmitate and an increased sensitivity to oxidative stress. Our results demonstrate that, even when NADPH is limiting, IDH1 mutants continue to synthesize 2-HG at the expense of other NADPH-requiring pathways that are essential for cell viability. Thus, rather than attempting to decrease 2-HG synthesis in the clinic, the consumption of NADPH by mutant IDH1 may be exploited as a metabolic weakness that sensitizes tumor cells to ionizing radiation, a commonly used anti-cancer therapy. : Using liquid chromatography/mass spectrometry (LC/MS and stable isotope tracing, Gelman et al. find that 2-HG production in cells with IDH1 mutations leads to increased pentose phosphate pathway activity to generate NADPH. Production of 2-HG competes with other NADPH-dependent pathways and sensitizes cells to redox stress. Keywords: 2-hydroxyglutarate, cancer metabolism, LC/MS, metabolomcis, pentose phosphate pathway, redox regulation

NADPH Oxidases: Progress and Opportunities

OpenAIRE

San Martin, Alejandra; Griendling, Kathy K.

2014-01-01

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

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

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Quan-Guang Zhang

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

NADPH promotes the rapid growth of the tumor

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Hao Sheng

2018-04-01

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

The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice.

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Rezende, Flávia; Moll, Franziska; Walter, Maria; Helfinger, Valeska; Hahner, Fabian; Janetzko, Patrick; Ringel, Christian; Weigert, Andreas; Fleming, Ingrid; Weissmann, Norbert; Kuenne, Carsten; Looso, Mario; Rieger, Michael A; Nawroth, Peter; Fleming, Thomas; Brandes, Ralf P; Schröder, Katrin

2018-05-01

NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes. In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice. ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Modulation of NADPH oxidase activity by known uraemic retention solutes

DEFF Research Database (Denmark)

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

2014-01-01

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

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

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Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

2014-08-01

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Modulation of NADPH oxidase activity by known uraemic retention solutes.

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

2014-08-01

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

NADPH oxidase: an enzyme for multicellularity?

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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.

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

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Philip E Morgan

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

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

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

1987-01-01

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

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

International Nuclear Information System (INIS)

Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

2014-01-01

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b 5 , and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b 5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b 5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b 5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

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

International Nuclear Information System (INIS)

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

2015-01-01

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D_2 (PGD_2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP"+, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-12-04

Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

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

Directory of Open Access Journals (Sweden)

Simona-Adriana Manea

2018-06-01

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

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

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Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

2011-08-01

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Effects of physical characteristics of carbon black on metabolic regulation in mice

International Nuclear Information System (INIS)

Chuang, Hsiao-Chi; Hsiao, Ta-Chih; Lee, Chii-Hong; Chun-Te Lin, Justin; Chuang, Kai-Jen; Feng, Po-Hao; Cheng, Tsun-Jen

2018-01-01

Potential adverse effects of human exposure to carbon black (CB) have been reported, but limited knowledge regarding CB-regulated metabolism is currently available. To evaluate how physical parameters of CB influence metabolism, we investigated CB and diesel exhaust particles (DEPs) and attempted to relate various physical parameters, including the hydrodynamic diameter, zeta potential, and particle number concentrations, to lung energy metabolism in female BALB/c mice. A body weight increase was arrested by 3 months of exposure to CB of smaller-size fractions, which was negatively correlated with pyruvate in plasma. There were no significant differences in cytotoxic lactate dehydrogenase (LDH) or total protein in bronchoalveolar lavage fluid (BALF) after 3 months of CB exposure. However, we observed alterations in acetyl CoA and the NADP/NADPH ratio in lung tissues with CB exposure. Additionally, the NADP/NADPH ratio was associated with the zeta potential of CB. Mild peribronchiovascular and interstitial inflammation and multinucleated giant cells (macrophages) with a transparent and rhomboid appearance and containing foreign bodies were observed in lung sections. We suggest that physical characteristics of CB, such as the zeta potential, may disrupt metabolism after pulmonary exposure. These results, therefore, provide the first evidence of a link between pulmonary exposure to CB and metabolism. - Highlights: • We evaluated how physical parameters of CB influence metabolism in mice lungs. • Body weight was arrested by exposure to CB in mice. • CB-induced peribronchiovascular and interstitial inflammation in the lungs. • Disruption in functions of acetyl CoA and NADP/NADPH was observed in lungs. • NADP/NADPH ratio was associated with the zeta potentials of CB.

Mitochondrial type II NAD(PH dehydrogenases in fungal cell death

Directory of Open Access Journals (Sweden)

A. Pedro Gonçalves

2015-03-01

Full Text Available During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(PH dehydrogenases (also called alternative NAD(PH dehydrogenases are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(PH dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(PH dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF-family.

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

Science.gov (United States)

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

Science.gov (United States)

Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

2018-04-03

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

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

Science.gov (United States)

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

2017-07-01

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

Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes

International Nuclear Information System (INIS)

Butterick, C.J.; Baehner, R.L.; Boxer, L.A.; Jersild, R.A. Jr.

1983-01-01

The cellular sites of H 2 O 2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1- 14 C oxidation to 14 CO 2 . Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H 2 O 2 in the phagocytizing granulocyte

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

Science.gov (United States)

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

2004-10-01

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

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

Directory of Open Access Journals (Sweden)

Luiza A. Rabêlo

2010-05-01

Full Text Available Vários estudos destacam as espécies reativas de oxigênio e nitrogênio (ERONs como importantes contribuintes na patogênese de numerosas doenças cardiovasculares, incluindo hipertensão, aterosclerose e falência cardíaca. Tais espécies são moléculas altamente bioativas e com vida curta derivadas, principalmente, da redução do oxigênio molecular. O complexo enzimático da NADPH oxidase é a maior fonte dessas espécies reativas na vasculatura. Sob condições fisiológicas, a formação e eliminação destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanço redox, entretanto, há um aumento na atividade da NADPH oxidase e predomínio de agentes pró-oxidantes, superando a capacidade de defesa orgânica antioxidante. Além disso, tal hiperatividade enzimática reduz a biodisponibilidade do óxido nítrico, crucial para a vasodilatação e a manutenção da função vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente à disfunção endotelial, foi primeiramente descrita por sua expressão em fagócitos, onde sua atividade determina a eficácia dos mecanismos de defesa orgânica contra patógenos. As sutis diferenças existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicações terapêuticas, permitindo a inibição seletiva do desequilíbrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participação nas vias fisiológicas de sinalização celular que garantem a proteção contra microorganismos.Varios estudios destacan las especies reactivas de oxígeno y nitrógeno (ERON como importantes contribuyentes en la patogénesis de numerosas enfermedades cardiovasculares, incluyendo hipertensión, aterosclerosis y falla cardíaca. Tales especies son moléculas altamente bioactivas y con vida corta derivadas, principalmente, de la reducción del oxígeno molecular. El complejo enzimático de la NADPH oxidasa es

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

Science.gov (United States)

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

2009-04-01

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

Idh2 deficiency accelerates renal dysfunction in aged mice.

Science.gov (United States)

Lee, Su Jeong; Cha, Hanvit; Lee, Seoyoon; Kim, Hyunjin; Ku, Hyeong Jun; Kim, Sung Hwan; Park, Jung Hyun; Lee, Jin Hyup; Park, Kwon Moo; Park, Jeen-Woo

2017-11-04

The free radical or oxidative stress theory of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species (ROS) that are produced as by-products of normal metabolic processes in mitochondria. The oxidative stress may arise as a result of either increased ROS production or decreased ability to detoxify ROS. The availability of the mitochondrial NADPH pool is critical for the maintenance of the mitochondrial antioxidant system. The major enzyme responsible for generating mitochondrial NADPH is mitochondrial NADP + -dependent isocitrate dehydrogenase (IDH2). Depletion of IDH2 in mice (idh2 -/- ) shortens life span and accelerates the degeneration of multiple age-sensitive traits, such as hair grayness, skin pathology, and eye pathology. Among the various internal organs tested in this study, IDH2 depletion-induced acceleration of senescence was uniquely observed in the kidney. Renal function and structure were greatly deteriorated in 24-month-old idh2 -/- mice compared with wild-type. In addition, disruption of redox status, which promotes oxidative damage and apoptosis, was more pronounced in idh2 -/- mice. These data support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice, and thus support the oxidative stress theory of aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Sex-related differences in NADPH-dependent lipid peroxidation induced by cadmium

Energy Technology Data Exchange (ETDEWEB)

Sato, Masao; Nagai, Yasushi

1986-10-01

Male and female rats were dosed once a day for 2 days with injections of 1.5 mg Cd/kg. Formation of thiobarbituric acid reactive substances (TBA-RS) was significantly increased in male rat liver but not in the females. NADPH-dependent lipid peroxidation in vitro in microsomes derived from untreated rat liver was greater in males than in females. Furthermore, addition of cadmium (Cd) to microsomes isolated from male rat liver produced a dose-dependent potentiation of NADPH-dependent lipid peroxidation from low concentrations of CD. In microsomes derived from females a significant increase in lipid peroxidation was observed only at high Cd concentrations. NADPH-dependent lipid peroxidation enhanced by Cd was greater in the males than in the females. These data suggest that a sex-related difference in the ability of Cd to induce lipid peroxidation in vivo in rat liver appears to be mediated partly through differences in hepatic microsomal NADPH-dependent lipid peroxidation.

Influenza virus-induced alterations of cytochrome P-450 enzyme activities following exposure of mice to coal and diesel particulates.

Science.gov (United States)

Rabovsky, J; Judy, D J; Rodak, D J; Petersen, M

1986-06-01

We have investigated a relationship between two detoxication systems, metabolic detoxication through the cytochrome P-450 (P-450) pathway and resistance to infection through interferon (IFN), in mice infected with influenza virus following exposure to coal dust (CD) and diesel exhaust (DE) particulates. Mice were exposed by inhalation to filtered air (FA; control), CD, or DE for 1 month and then inoculated intranasally (IN) with influenza virus. During infection, 7-ethoxycoumarin deethylase (7ECdeEt'ase) and ethylmorphine demethylase (EMdeMe'ase) (monooxygenases), and NADPH cytochrome c reductase (NADPH c red'ase) were measured in liver microsomes. Temporal patterns of enzyme activities were observed with control animals. EMdeMe'ase and NADPH c red'ase exhibited peak values at Day 4 postinfection (27.6 and 482 nmole/min/mg protein, respectively), compared to initial activities (9.1 and 307 nmole/min/mg protein, respectively). 7ECdeEt'ase activity decreased between Days 1-3 postvirus infection and thereafter returned to the original value (1.7 nmole/min/mg protein). When the mice were first exposed to CD or DE particulates for 1 month prior to influenza infection, changes in enzyme temporal patterns were observed. The increased EMdeMe'ase activity at Day 4 was not observed in mice exposed to CD and was reduced in mice exposed to DE. Preexposure to either particulate resulted in the abolition of the increased Day 4 activity of NADPH c red'ase. The 7ECdeEt'ase postinfection temporal pattern was not affected by a preexposure to either particulate. Estimates of the enzyme activities after the 1-month exposure to FA, CD, or DE but before virus infection indicated no changes due to particulate exposure alone. Under these conditions of particulate exposure and virus infection, serum IFN levels in the mice used in this study peaked at Days 4-5 and were unaffected by the 1-month preexposure to CD or DE (Hahon et al., (1985). The data suggest the relationship that exists

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

Energy Technology Data Exchange (ETDEWEB)

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

2014-03-01

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

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

Science.gov (United States)

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.

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

NARCIS (Netherlands)

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

2011-01-01

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

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

Science.gov (United States)

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

2016-11-01

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

Pancreatic Beta-Cell Purification by Altering FAD and NAD(PH Metabolism

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P. de Vos

2008-07-01

Full Text Available Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD and nicotinamide-adenine dinucleotide phosphate (NAD(PH autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(PH fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(PH contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(PH fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

Engineering an NADPH/NADP⁺ Redox Biosensor in Yeast

DEFF Research Database (Denmark)

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

2016-01-01

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

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

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José Luis Miguel-Carrasco

2012-01-01

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

Influenza virus-induced alterations of cytochrome P-450 enzyme activities following exposure of mice to coal and diesel particulates

Energy Technology Data Exchange (ETDEWEB)

Rabovsky, J.; Judy, D.J.; Rodak, D.J.; Petersen, M.

1986-06-01

We have investigated a relationship between two detoxication systems, metabolic detoxication through the cytochrome P-450 (P-450) pathway and resistance to infection through interferon (IFN), in mice infected with influenza virus following exposure to coal dust (CD) and diesel exhaust (DE) particulates. Mice were exposed by inhalation to filtered air (FA; control), CD, or DE for 1 month and then inoculated intranasally (IN) with influenza virus. During infection, 7-ethoxycoumarin deethylase (7ECdeEt'ase) and ethylmorphine demethylase (EMdeMe'ase) (monooxygenases), and NADPH cytochrome c reductase (NADPH c red'ase) were measured in liver microsomes. Temporal patterns of enzyme activities were observed with control animals. EMdeMe'ase and NADPH c red'ase exhibited peak values at Day 4 postinfection (27.6 and 482 nmole/min/mg protein, respectively), compared to initial activities (9.1 and 307 nmole/min/mg protein, respectively). 7ECdeEt'ase activity decreased between Days 1-3 postvirus infection and thereafter returned to the original value (1.7 nmole/min/mg protein). When the mice were first exposed to CD or DE particulates for 1 month prior to influenza infection, changes in enzyme temporal patterns were observed. The increased EMdeMe'ase activity at Day 4 was not observed in mice exposed to CD and was reduced in mice exposed to DE. Preexposure to either particulate resulted in the abolition of the increased Day 4 activity of NADPH c red'ase. The 7ECdeEt'ase postinfection temporal pattern was not affected by a preexposure to either particulate. Estimates of the enzyme activities after the 1-month exposure to FA, CD, or DE but before virus infection indicated no changes due to particulate exposure alone. Under conditions of particulate exposure and virus infection, serum IFN levels peaked at Days 4-5 and were unaffected by the 1-month preexposure to CD or DE.

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

DEFF Research Database (Denmark)

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

1997-01-01

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

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

Science.gov (United States)

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.

NADPH-diaphorase expression in the Meibomian glands of rat palpebra in postnatal development

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D. Kluchova

2010-11-01

Full Text Available In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS positive nerve fibers in rat meibomian glands (MGs at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d is a surrogate for neuronal nitric oxide synthase (NOS. Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.

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

Science.gov (United States)

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

2003-01-01

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

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

Directory of Open Access Journals (Sweden)

Arango Rincón, Julián Camilo

2010-12-01

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

Silymarin alleviates hepatic oxidative stress and protects against metabolic disorders in high-fat diet-fed mice.

Science.gov (United States)

Feng, Bin; Meng, Ran; Huang, Bin; Shen, Shanmei; Bi, Yan; Zhu, Dalong

2016-01-01

Silymarin is a potent antioxidant medicine and has been widely used for the treatment of liver diseases over 30 years. Recent studies suggest that silymarin may benefit patients with glucose intolerance. However, the mechanism underlying the action of silymarin is not clarified. The aim of this work was to assess the impact of silymarin on glucose intolerance in high-fat diet (HFD)-fed mice, and explore the potential therapeutic mechanisms. C57BL/6 mice were fed with HFD for 12 weeks, randomized, and treated orally with vehicle saline or silymarin (30 mg/kg) daily for 30 days. We found that silymarin significantly improved HFD-induced body weight gain, glucose intolerance, and insulin resistance in mice. Silymarin treatment reduced HFD-increased oxidative stress indicators (reactive oxygen species, lipid peroxidation, protein oxidation) and restored HFD-down-regulated activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) in the plasma and/or liver of the HFD-fed mice. Furthermore, silymarin decreased HFD-up-regulated hepatic NADPH oxidase expression and NF-κB activation in mice. Additionally, silymarin treatment mitigated HFD-increased plasma IL-1β, TNF-α levels, and HFD-enhanced hepatic NO, TLR4, and iNOS expression in mice. These novel data indicate that silymarin has potent anti-diabetic actions through alleviating oxidative stress and inflammatory response, partially by inhibiting hepatic NADPH oxidase expression and the NF-κB signaling.

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

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Guillermo Bodega

2018-01-01

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

TAM Receptors Are Not Required for Zika Virus Infection in Mice.

Science.gov (United States)

Hastings, Andrew K; Yockey, Laura J; Jagger, Brett W; Hwang, Jesse; Uraki, Ryuta; Gaitsch, Hallie F; Parnell, Lindsay A; Cao, Bin; Mysorekar, Indira U; Rothlin, Carla V; Fikrig, Erol; Diamond, Michael S; Iwasaki, Akiko

2017-04-18

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl -/- , Mertk -/- , Axl -/- Mertk -/- , and Axl -/- Tyro3 -/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-30

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

Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

Science.gov (United States)

Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

1976-03-01

Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-05-01

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

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

International Nuclear Information System (INIS)

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

2017-01-01

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

Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

International Nuclear Information System (INIS)

Kirkensgaard, Kristine G.; Hägglund, Per; Finnie, Christine; Svensson, Birte; Henriksen, Anette

2009-01-01

The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R cryst of 19.0% and an R free of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation

Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

Energy Technology Data Exchange (ETDEWEB)

Kirkensgaard, Kristine G. [Carlsberg Laboratory (Denmark); Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Hägglund, Per; Finnie, Christine; Svensson, Birte [Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Henriksen, Anette, E-mail: anette@crc.dk [Carlsberg Laboratory (Denmark)

2009-09-01

The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.

Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

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Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

2015-12-01

Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

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Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

2005-01-28

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

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

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Chertok, V M; Kotsuba, A E

2013-01-01

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

Chronic apocynin treatment attenuates beta amyloid plaque size and microglial number in hAPP(751(SL mice.

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Melinda E Lull

Full Text Available NADPH oxidase is implicated in neurotoxic microglial activation and the progressive nature of Alzheimer's Disease (AD. Here, we test the ability of two NADPH oxidase inhibitors, apocynin and dextromethorphan (DM, to reduce learning deficits and neuropathology in transgenic mice overexpressing human amyloid precursor protein with the Swedish and London mutations (hAPP(751(SL.Four month old hAPP(751(SL mice were treated daily with saline, 15 mg/kg DM, 7.5 mg/kg DM, or 10 mg/kg apocynin by gavage for four months.Only hAPP(751(SL mice treated with apocynin showed reduced plaque size and a reduction in the number of cortical microglia, when compared to the saline treated group. Analysis of whole brain homogenates from all treatments tested (saline, DM, and apocynin demonstrated low levels of TNFα, protein nitration, lipid peroxidation, and NADPH oxidase activation, indicating a low level of neuroinflammation and oxidative stress in hAPP(751(SL mice at 8 months of age that was not significantly affected by any drug treatment. Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751(SL mice. To discern how apocynin was affecting plaque levels (plaque load and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ phagocytosis, microglial proliferation, or microglial survival.Together, this study suggests that while hAPP(751(SL mice show increases in microglial number and plaque load, they fail to exhibit elevated markers of neuroinflammation consistent with AD at 8 months of age, which may be a limitation of this animal model. Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional

TAM Receptors Are Not Required for Zika Virus Infection in Mice

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Andrew K. Hastings

2017-04-01

Full Text Available Summary: Tyro3, Axl, and Mertk (TAM receptors are candidate entry receptors for infection with the Zika virus (ZIKV, an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl−/−, Mertk−/−, Axl−/−Mertk−/−, and Axl−/−Tyro3−/− mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR-blocking (MAR1-5A3 antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection. : TAM receptors have been implicated as entry receptors for the Zika virus. In this study, Hastings et al. used genetic knockout mouse models to demonstrate that they are not necessary for the infection of mice via multiple routes of viral challenge. These results suggest the existence of redundant entry receptors for ZIKV in mice. Keywords: viral entry, flavivirus, neurotropic virus, CNS, pregnancy, congenital infection

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

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

2015-09-15

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

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

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

2009-10-01

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

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

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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.

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

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Gábor Sirokmány

2018-06-01

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

Nox-2-mediated phenotype loss of hippocampal parvalbumin interneurons might contribute to postoperative cognitive decline in aging mice

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lili qiu

2016-10-01

Full Text Available Postoperative cognitive decline (POCD is a common complication following anesthesia and surgery, especially in elderly patients; however, the precise mechanisms of POCD remain unclear. Here, we investigated whether nicotinamide adenine dinucleotide phosphate (NADPH oxidase mediated-abnormalities in parvalbumin (PV interneurons play an important role in the pathophysiology of POCD. The animal model was established using isoflurane anesthesia and exploratory laparotomy in sixteen-month-old male C57BL/6 mice. For interventional experiments, mice were chronically treated with the NADPH oxidase inhibitor apocynin (APO. Open field and fear conditioning behavioral tests were performed on day 6 and 7 post-surgery, respectively. In a separate experiment, brain tissue was harvested and subjected to biochemical analysis. Primary hippocampal neurons challenged with lipopolysaccharide in vitro were used to investigate the mechanisms underlying the oxidative stress-induced abnormalities in PV interneurons. Our results showed that anesthesia and surgery induced significant hippocampus-dependent memory impairment, which was accompanied by PV interneuron phenotype loss and increased expression of interleukin-1β, markers of oxidative stress, and NADPH oxidase 2 (Nox2 in the hippocampus. In addition, lipopolysaccharide exposure increased Nox2 level and decreased the expression of PV and the number of excitatory synapses onto PV interneurons in the primary hippocampal neurons. Notably, treatment with APO reversed these abnormalities. Our study suggests that Nox2-derived ROS production triggers, at least in part, anesthesia- and surgery-induced hippocampal PV interneuron phenotype loss and consequent cognitive impairment in aging mice.

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

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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.

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

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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.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

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Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2013-11-01

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

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Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

2018-01-01

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

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Katarína Smolková

2012-01-01

Full Text Available Isocitrate dehydrogenase 2 (IDH2 is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+ accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction, which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α.

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

International Nuclear Information System (INIS)

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

2011-01-01

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O 3 ) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O 3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O 3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O 3 , determined from the mRNA levels of the major allergens. We conclude that O 3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: → O 3 reduces the viability of ragweed pollen. → ROS and allergens of ragweed pollen were not affected by O 3 exposure. → O 3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. → O 3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

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

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

2011-10-15

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O{sub 3} fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

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

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F. Dorko

2012-02-01

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

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

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Ashutosh Kumar

2018-05-01

Full Text Available Exposure to (bisulfite (HSO3– and sulfite (SO32– has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bisulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–, peroxymonosulfate (–O3SOO., and especially the sulfate (SO4. – anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO, which has been shown to form protein radicals. Although formation of (bisulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bisulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bisulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bisulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bisulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bisulfite-derived protein radical formation and show the involvement of

Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

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Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

2013-11-08

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

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

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Victoria Campuzano

2012-02-01

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

Purification, crystallization and preliminary X-ray analysis of l-sorbose reductase from Gluconobacter frateurii complexed with l-sorbose or NADPH

International Nuclear Information System (INIS)

Kubota, Keiko; Nagata, Koji; Miyazono, Ken-ichi; Toyama, Hirohide; Matsushita, Kazunobu; Tanokura, Masaru

2009-01-01

NADPH-dependent l-sorbose reductase from G. frateurii (SR) was expressed, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method. Crystals of the SR–l-sorbose complex and SR–NADPH complex diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. NADPH-dependent l-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR–l-sorbose complex and the SR–NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. The crystal of the SR–l-sorbose complex belonged to space group C222 1 , with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 Å. The crystal of the SR–NADPH complex belonged to space group P2 1 , with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 Å, β = 89.99°. The crystals contained two and eight molecules, respectively, in the asymmetric unit

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

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

2015-05-01

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

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

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

2014-01-01

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

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

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Alberto eGonzález

2014-10-01

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

Leptin Is Required for Glucose Homeostasis after Roux-en-Y Gastric Bypass in Mice.

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Mohamad Mokadem

Full Text Available Leptin, the protein product of the ob gene, increases energy expenditure and reduces food intake, thereby promoting weight reduction. Leptin also regulates glucose homeostasis and hepatic insulin sensitivity via hypothalamic proopiomelanocortin neurons in mice. Roux-en-Y gastric bypass (RYGB induces weight loss that is substantial and sustained despite reducing plasma leptin levels. In addition, patients who fail to undergo diabetes remission after RYGB are hypoletinemic compared to those who do and to lean controls. We have previously demonstrated that the beneficial effects of RYGB in mice require the melanocortin-4 receptor, a downstream effector of leptin action. Based on these observations, we hypothesized that leptin is required for sustained weight reduction and improved glucose homeostasis observed after RYGB.To investigate this hypothesis, we performed RYGB or sham operations on leptin-deficient ob/ob mice maintained on regular chow. To investigate whether leptin is involved in post-RYGB weight maintenance, we challenged post-surgical mice with high fat diet.RYGB reduced total body weight, fat and lean mass and caused reduction in calorie intake in ob/ob mice. However, it failed to improve glucose tolerance, glucose-stimulated plasma insulin, insulin tolerance, and fasting plasma insulin. High fat diet eliminated the reduction in calorie intake observed after RYGB in ob/ob mice and promoted weight regain, although not to the same extent as in sham-operated mice. We conclude that leptin is required for the effects of RYGB on glucose homeostasis but not body weight or composition in mice. Our data also suggest that leptin may play a role in post-RYGB weight maintenance.

NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

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Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

2018-01-20

The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

Interferon gamma/NADPH oxidase defence system in immunity and cancer

Czech Academy of Sciences Publication Activity Database

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

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

Podocyte specific knock out of selenoproteins does not enhance nephropathy in streptozotocin diabetic C57BL/6 mice

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Carlson Bradley A

2008-07-01

Full Text Available Abstract Background Selenoproteins contain selenocysteine (Sec, commonly considered the 21st genetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/or through their roles in the maintenance of intracellular redox balance. Since oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteins protect against this complication of diabetes. Methods C57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins (denoted in this paper as PodoTrsp-/- and control mice were made diabetic by intraperitoneal injection of streptozotocin, or were injected with vehicle. Blood glucose, body weight, microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers of oxidative stress were assessed. Results After 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of blood glucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff staining to examine mesangial matrix expansion also demonstrated no difference between control and PodoTrsp-/- mice after 6 months of diabetes, and there were no differences in immunohistochemical stainings for nitrotyrosine or NAD(PH dehydrogenase, quinone 1. Conclusion Loss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does not lead to increased oxidative stress as assessed by nitrotyrosine and NAD(PH dehydrogenase, quinone 1 immunostaining, nor does it lead to worsening nephropathy.

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

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Gi Soo Youn

2017-08-01

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

A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network.

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Ranji Singh

Full Text Available The reduced nicotinamide adenine dinucleotide phosphate (NADPH is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH, a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC, malic enzyme (ME, malate dehydrogenase (MDH, malate synthase (MS, and isocitrate lyase (ICL that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK and the upregulation of pyruvate kinase (PK ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.

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

DEFF Research Database (Denmark)

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

2008-01-01

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

[Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803].

Science.gov (United States)

Xie, Juan; Zhou, Jie; Zhang, Haifeng; Li, Yin

2011-07-01

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.

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

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Jagna Chmielowska-Bąk

2017-06-01

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

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

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Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-05-01

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

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Crankshaw, D L; Hetnarski, K; Wilkinson, C F

1979-09-01

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

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Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

African Journals Online (AJOL)

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

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

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Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

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

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

2014-03-01

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

Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

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Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

2016-06-01

Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

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Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2014-01-01

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

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

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Landreth Gary E

2006-11-01

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

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

International Nuclear Information System (INIS)

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

2008-01-01

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

(--Epigallocatechin gallate attenuates NADPH-d/nNOS expression in motor neurons of rats following peripheral nerve injury

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Tseng Chi-Yu

2011-06-01

Full Text Available Abstract Background Oxidative stress and large amounts of nitric oxide (NO have been implicated in the pathophysiology of neuronal injury and neurodegenerative disease. Recent studies have shown that (--epigallocatechin gallate (EGCG, one of the green tea polyphenols, has potent antioxidant effects against free radical-mediated lipid peroxidation in ischemia-induced neuronal damage. The purpose of this study was to examine whether EGCG would attenuate neuronal expression of NADPH-d/nNOS in the motor neurons of the lower brainstem following peripheral nerve crush. Thus, young adult rats were treated with EGCG (10, 25, or 50 mg/kg, i.p. 30 min prior to crushing their hypoglossal and vagus nerves for 30 seconds (left side, at the cervical level. The treatment (pre-crush doses of EGCG was continued from day 1 to day 6, and the animals were sacrificed on days 3, 7, 14 and 28. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS immunohistochemistry were used to assess neuronal NADPH-d/nNOS expression in the hypoglossal nucleus and dorsal motor nucleus of the vagus. Results In rats treated with high dosages of EGCG (25 or 50 mg/kg, NADPH-d/nNOS reactivity and cell death of the motor neurons were significantly decreased. Conclusions The present evidence indicated that EGCG can reduce NADPH-d/nNOS reactivity and thus may enhance motor neuron survival time following peripheral nerve injury.

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

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Marco Aurelio M Freire

2008-05-01

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

Mitochondrial NAD(PH in vivo: identifying natural indicators of oxidative phosphorylation in the 31P magnetic resonance spectrum.

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Kevin eConley

2016-03-01

Full Text Available Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P+ and NAD(PH, which are compartmentalized between cell and mitochondria. Here we provide evidence for detection of NAD(P+ and NAD(PH in separate mitochondrial and cell pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy (31P MRS. These NAD(P pools are identified by chemical standards (NAD+, NADP+ and NADH and by physiological tests. A unique resonance reflecting mitochondrial NAD(PH is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(PH with oxidation is matched by a stoichiometric rise in the NAD(P+ peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(PH peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus non-invasive detection of NAD(P+ and NAD(PH in cell vs. mitochondria yield natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

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

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

2011-10-01

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

International Nuclear Information System (INIS)

Uehleke, H.; Poplawski-Tabarelli, S.

1977-01-01

1) 14 C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h : 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein. 2) The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extraction removed 60-70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital. (orig.) [de

SEC23B is required for pancreatic acinar cell function in adult mice

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Khoriaty, Rami; Vogel, Nancy; Hoenerhoff, Mark J.; Sans, M. Dolors; Zhu, Guojing; Everett, Lesley; Nelson, Bradley; Durairaj, Haritha; McKnight, Brooke; Zhang, Bin; Ernst, Stephen A.; Ginsburg, David; Williams, John A.

2017-01-01

Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell–specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice. PMID:28539403

NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

NARCIS (Netherlands)

Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

2016-01-01

NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

Science.gov (United States)

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

2016-02-01

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

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

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

2012-06-22

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

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

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

2017-08-01

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

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

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

2018-04-01

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

Transhydrogenase promotes the robustness and evolvability of E. coli deficient in NADPH production.

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Hsin-Hung Chou

Full Text Available Metabolic networks revolve around few metabolites recognized by diverse enzymes and involved in myriad reactions. Though hub metabolites are considered as stepping stones to facilitate the evolutionary expansion of biochemical pathways, changes in their production or consumption often impair cellular physiology through their system-wide connections. How does metabolism endure perturbations brought immediately by pathway modification and restore hub homeostasis in the long run? To address this question we studied laboratory evolution of pathway-engineered Escherichia coli that underproduces the redox cofactor NADPH on glucose. Literature suggests multiple possibilities to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from our twelve evolved populations revealed merely two solutions: (1 modulating the expression of membrane-bound transhydrogenase (mTH in every population; (2 simultaneously consuming glucose with acetate, an unfavored byproduct normally excreted during glucose catabolism, in two subpopulations. Notably, mTH displays broad phylogenetic distribution and has also played a predominant role in laboratory evolution of Methylobacterium extorquens deficient in NADPH production. Convergent evolution of two phylogenetically and metabolically distinct species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification via evolving dual substrate consumption highlights the flexibility of physiological systems to exploit ecological opportunities.

Transhydrogenase Promotes the Robustness and Evolvability of E. coli Deficient in NADPH Production

Science.gov (United States)

Chou, Hsin-Hung; Marx, Christopher J.; Sauer, Uwe

2015-01-01

Metabolic networks revolve around few metabolites recognized by diverse enzymes and involved in myriad reactions. Though hub metabolites are considered as stepping stones to facilitate the evolutionary expansion of biochemical pathways, changes in their production or consumption often impair cellular physiology through their system-wide connections. How does metabolism endure perturbations brought immediately by pathway modification and restore hub homeostasis in the long run? To address this question we studied laboratory evolution of pathway-engineered Escherichia coli that underproduces the redox cofactor NADPH on glucose. Literature suggests multiple possibilities to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from our twelve evolved populations revealed merely two solutions: (1) modulating the expression of membrane-bound transhydrogenase (mTH) in every population; (2) simultaneously consuming glucose with acetate, an unfavored byproduct normally excreted during glucose catabolism, in two subpopulations. Notably, mTH displays broad phylogenetic distribution and has also played a predominant role in laboratory evolution of Methylobacterium extorquens deficient in NADPH production. Convergent evolution of two phylogenetically and metabolically distinct species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification via evolving dual substrate consumption highlights the flexibility of physiological systems to exploit ecological opportunities. PMID:25715029

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

Science.gov (United States)

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

2016-01-01

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

Quench Protection and Magnet Powe Supply Requirements for the MICE Focusing and Coupling Magnets

International Nuclear Information System (INIS)

Green, Michael A.; Witte, Holger

2005-01-01

This report discusses the quench protection and power supply requirements of the MICE superconducting magnets. A section of the report discusses the quench process and how to calculate the peak voltages and hotspot temperature that result from a magnet quench. A section of the report discusses conventional quench protection methods. Thermal quench back from the magnet mandrel is also discussed. Selected quench protection methods that result in safe quenching of the MICE focusing and coupling magnets are discussed. The coupling of the MICE magnets with the other magnets in the MICE is described. The consequences of this coupling on magnet charging and quenching are discussed. Calculations of the quenching of a magnet due quench back from circulating currents induced in the magnet mandrel due to quenching of an adjacent magnet are discussed. The conclusion of this report describes how the MICE magnet channel will react when one or magnets in that channel are quenched

Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

Science.gov (United States)

Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

2018-01-01

The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2017-05-01

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

Crystallization and preliminary X-ray analysis of 5-keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-d-gluconate and NADPH

International Nuclear Information System (INIS)

Kubota, Keiko; Miyazono, Ken-ichi; Nagata, Koji; Toyama, Hirohide; Matsushita, Kazunobu; Tanokura, Masaru

2010-01-01

NADPH-dependent 5-keto-d-gluconate reductase from G. suboxydans IFO12528 (5KGR) was expressed, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method. Crystals of the 5KGR–NADPH complex and of the 5KGR–NADPH–5-keto-d-gluconate complex diffracted X-rays to 1.75 and 2.26 Å resolution, respectively. NADPH-dependent 5-keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-d-gluconate and d-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR–NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Å resolution. The crystal of the complex belonged to space group P4 2 2 1 2, with unit-cell parameters a = b = 128.6, c = 62.9 Å. A crystal of the 5KGR–NADPH–5-keto-d-gluconate complex was prepared by soaking the 5KGR–NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-d-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P4 2 2 1 2, with unit-cell parameters a = b = 128.7, c = 62.5 Å. Both crystals contained two molecules in the asymmetric unit

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

Science.gov (United States)

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

2017-02-01

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

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

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Giang T. Nguyen

2017-08-01

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

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

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

2015-01-20

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

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

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

2015-10-01

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

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice

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Li, Zhenzu; Zhang, Tingting; Lin, Zhuchun; Hou, Congzhe; Zhang, Jian; Men, Yuqin; Li, Huashun

2016-01-01

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice. PMID:27603780

NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

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

2014-01-01

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

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

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Koji Mizuhashi

Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

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Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

2015-01-01

Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

DEFF Research Database (Denmark)

Panagiotou, Gianni; Christakopoulos, P.

2004-01-01

Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...

Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

DEFF Research Database (Denmark)

Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

2011-01-01

The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR und...... to serve as an effective electron transferring "nano-machine"....

Direct renin inhibitor ameliorates insulin resistance by improving insulin signaling and oxidative stress in the skeletal muscle from post-infarct heart failure in mice.

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Fukushima, Arata; Kinugawa, Shintaro; Takada, Shingo; Matsumoto, Junichi; Furihata, Takaaki; Mizushima, Wataru; Tsuda, Masaya; Yokota, Takashi; Matsushima, Shouji; Okita, Koichi; Tsutsui, Hiroyuki

2016-05-15

Insulin resistance can occur as a consequence of heart failure (HF). Activation of the renin-angiotensin system (RAS) may play a crucial role in this phenomenon. We thus investigated the effect of a direct renin inhibitor, aliskiren, on insulin resistance in HF after myocardial infarction (MI). MI and sham operation were performed in male C57BL/6J mice. The mice were divided into 4 groups and treated with sham-operation (Sham, n=10), sham-operation and aliskiren (Sham+Aliskiren; 10mg/kg/day, n=10), MI (n=11), or MI and aliskiren (MI+Aliskiren, n=11). After 4 weeks, MI mice showed left ventricular dilation and dysfunction, which were not affected by aliskiren. The percent decrease of blood glucose after insulin load was significantly smaller in MI than in Sham (14±5% vs. 36±2%), and was ameliorated in MI+Aliskiren (34±5%) mice. Insulin-stimulated serine-phosphorylation of Akt and glucose transporter 4 translocation were decreased in the skeletal muscle of MI compared to Sham by 57% and 69%, and both changes were ameliorated in the MI+Aliskiren group (91% and 94%). Aliskiren administration in MI mice significantly inhibited plasma renin activity and angiotensin II (Ang II) levels. Moreover, (pro)renin receptor expression and local Ang II production were upregulated in skeletal muscle from MI and were attenuated in MI+Aliskiren mice, in tandem with a decrease in superoxide production and NAD(P)H oxidase activities. In conclusion, aliskiren ameliorated insulin resistance in HF by improving insulin signaling in the skeletal muscle, at least partly by inhibiting systemic and (pro)renin receptor-mediated local RAS activation, and subsequent NAD(P)H oxidase-induced oxidative stress. Copyright © 2016 Elsevier B.V. All rights reserved.

Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

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Bhushan, Bharat; Halasz, Annamaria; Hawari, Jalal

2005-01-01

A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24 nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393 Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1 Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D - ) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H

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

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

2010-01-01

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

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

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Chin-Chung eLin

2016-03-01

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

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

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

2008-07-01

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

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

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Peiying Yu

2014-01-01

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

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

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Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

2013-11-01

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

The effects of Urtica dioica L. leaf extract on aniline 4-hydroxylase in mice.

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Ozen, Tevfik; Korkmaz, Halil

2009-01-01

The effects of hydroalcoholic (80% ethanol-20% water) extract of Urtica dioica L. on microsomal aniline 4-hydroxylase (A4H) were investigated in the liver of Swiss albino mice (8- 10-weeks-old) treated with two doses (50 and 100 mg/kg body weight, given orally for 14 days ). The activities of A4H showed a significant increase in the liver at both dose levels of extract treatment. The hydroalcoholic extract of Urtica dioica induced the activities of A4H that had been increased by treatment of metal ions (Mg2+ and Ca2+) and the mixture of cofactors (NADH and NADPH). At saturated concentration of cofactor, microsomal A4H exhibited significantly even higher activities in the presence of the mixture of cofactors than NADPH and NADH. Mg2+ and Ca2+ ions acted as stimulants in vitro. The present results suggest that the hydroalcoholic extract of Urtica dioica may have modalatory effect on aniline hydroxylase at least in part and enhance the activity of A4H adding metals ions and cofactors.

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

Science.gov (United States)

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was

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

Science.gov (United States)

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

2016-11-01

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

Structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

International Nuclear Information System (INIS)

Yajima, Shunsuke; Hara, Kodai; Iino, Daisuke; Sasaki, Yasuyuki; Kuzuyama, Tomohisa; Ohsawa, Kanju; Seto, Haruo

2007-01-01

The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg 2+ was shifted by NADPH in the active site

Antihypertensive effect of etamicastat in dopamine D2 receptor-deficient mice.

Science.gov (United States)

Armando, Ines; Asico, Laureano D; Wang, Xiaoyan; Jones, John E; Serrão, Maria Paula; Cuevas, Santiago; Grandy, David K; Soares-da-Silva, Patricio; Jose, Pedro A

2018-04-13

Abnormalities of the D 2 R gene (DRD2) play a role in the pathogenesis of human essential hypertension; variants of the DRD2 have been reported to be associated with hypertension. Disruption of Drd2 (D 2 -/- ) in mice increases blood pressure. The hypertension of D 2 -/- mice has been related, in part, to increased sympathetic activity, renal oxidative stress, and renal endothelin B receptor (ETBR) expression. We tested in D 2 -/- mice the effect of etamicastat, a reversible peripheral inhibitor of dopamine-β-hydroxylase that reduces the biosynthesis of norepinephrine from dopamine and decreases sympathetic nerve activity. Blood pressure was measured in anesthetized D 2 -/- mice treated with etamicastat by gavage, (10 mg/kg), conscious D 2 -/- mice, and D 2 +/+ littermates, and mice with the D 2 R selectively silenced in the kidney, treated with etamicastat in the drinking water (10 mg/kg per day). Tissue and urinary catecholamines and renal expression of selected G protein-coupled receptors, enzymes related to the production of reactive oxygen species, and sodium transporters were also measured. Etamicastat decreased blood pressure both in anesthetized and conscious D 2 -/- mice and mice with renal-selective silencing of D 2 R to levels similar or close to those measured in D 2 +/+ littermates. Etamicastat decreased cardiac and renal norepinephrine and increased cardiac and urinary dopamine levels in D 2 -/- mice. It also normalized the increased renal protein expressions of ETBR, NADPH oxidase isoenzymes, and urinary 8-isoprostane, as well as renal NHE3 and NCC, and increased the renal expression of D 1 R but not D 5 R in D 2 -/- mice. In conclusion, etamicastat is effective in normalizing the increased blood pressure and some of the abnormal renal biochemical alterations of D 2 -/- mice.

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

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María A. Hidalgo

2015-01-01

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

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

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

2016-02-01

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

Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

International Nuclear Information System (INIS)

Morrison, J.F.; Stone, S.R.

1988-01-01

The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/K/sub NADPH/ profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the K/sub i/ profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/K/sub NADPH/, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. The data for deuterium isotope and deuterium solvent isotope effects are consistent with the postulate that, for the reduction of dihydrofolate to tetrahydrofolate, protonation precedes hydride transfer. A scheme is proposed for the indirect transfer of a proton from the enzyme to dihydrofolate

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

OpenAIRE

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

1999-01-01

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

Increase in intracellular free/bound NAD[P]H as a cause of Cd-induced oxidative stress in the HepG2 cells

International Nuclear Information System (INIS)

Yang, M.S.; Li, D.; Lin, T.; Zheng, J.J.; Zheng, W.; Qu, J.Y.

2008-01-01

The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG 2 cells at 0.5, 1.5, 3 and 4.5 h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5 h after treatment while the change in FAD/NAD[P]H did not occur until 3 h after exposure. Production of ROS was also increased at 1.5 h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5 h and remained elevated until 4.5 h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events

Increasing Muscle Mass Improves Vascular Function in Obese (db/db) Mice

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Qiu, Shuiqing; Mintz, James D.; Salet, Christina D.; Han, Weihong; Giannis, Athanassios; Chen, Feng; Yu, Yanfang; Su, Yunchao; Fulton, David J.; Stepp, David W.

2014-01-01

Background A sedentary lifestyle is an independent risk factor for cardiovascular disease and exercise has been shown to ameliorate this risk. Inactivity is associated with a loss of muscle mass, which is also reversed with isometric exercise training. The relationship between muscle mass and vascular function is poorly defined. The aims of the current study were to determine whether increasing muscle mass by genetic deletion of myostatin, a negative regulator of muscle growth, can influence vascular function in mesenteric arteries from obese db/db mice. Methods and Results Myostatin expression was elevated in skeletal muscle of obese mice and associated with reduced muscle mass (30% to 50%). Myostatin deletion increased muscle mass in lean (40% to 60%) and obese (80% to 115%) mice through increased muscle fiber size (PMyostatin deletion decreased adipose tissue in lean mice, but not obese mice. Markers of insulin resistance and glucose tolerance were improved in obese myostatin knockout mice. Obese mice demonstrated an impaired endothelial vasodilation, compared to lean mice. This impairment was improved by superoxide dismutase mimic Tempol. Deletion of myostatin improved endothelial vasodilation in mesenteric arteries in obese, but not in lean, mice. This improvement was blunted by nitric oxide (NO) synthase inhibitor l‐NG‐nitroarginine methyl ester (l‐NAME). Prostacyclin (PGI2)‐ and endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilation were preserved in obese mice and unaffected by myostatin deletion. Reactive oxygen species) was elevated in the mesenteric endothelium of obese mice and down‐regulated by deletion of myostatin in obese mice. Impaired vasodilation in obese mice was improved by NADPH oxidase inhibitor (GKT136901). Treatment with sepiapterin, which increases levels of tetrahydrobiopterin, improved vasodilation in obese mice, an improvement blocked by l‐NAME. Conclusions Increasing muscle mass by genetic deletion of

Neurodegenerative evidence in mice brains with cecal ligation and puncture-induced sepsis: preventive effect of the free radical scavenger edaravone.

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Hiroki Yokoo

Full Text Available Sepsis is a major clinical challenge and septic encephalopathy is its nasty complication. The pathogenesis and underlying mechanisms of septic encephalopathy are not well understood. This study sought to fully characterize sepsis-associated biochemical and histopathological changes in brains of mice after cecal ligation and puncture, regarded as a highly clinically relevant animal model of polymicrobial sepsis. Real-time PCR analysis showed that gene expression levels of proinflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, were significantly up-regulated in brain tissues from septic mice, but to a much lesser extent when compared with those in peripheral tissues such as lungs. Blood-brain barrier (BBB permeability was significantly increased in septic mice, as determined by the measurement of sodium fluorescein and Evans blue content. Sepsis resulted in increases in NADPH oxidase activity and expression of p47(phox and p67(phox and up-regulation of inducible nitric oxide (NO synthase in brains, indicating that superoxide, produced by NADPH oxidase, reacts with NO to form peroxynitrite, that maybe lead to the loss of BBB integrity. Light and electron microscopic examination of septic mouse brain showed serious neuronal degeneration, as indicated by hyperchromatic, shrunken, pyknotic, and electron-dense neurons. These histopathological changes were prevented by treatment with the free radical scavenger edaravone. Together, these results suggest that sepsis can lead to rapid neurodegenerative changes in brains via free radical species production and possibly subsequent injury to the BBB. We may also provide a potentially useful therapeutic tool for treating septic encephalopathy.

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

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Stasia, Marie-José

2007-05-01

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

ONLINE MONITORING OF EXTRACELLULAR BRAIN GLUCOSE USING MICRODIALYSIS AND A NADPH-LINKED ENZYMATIC ASSAY

NARCIS (Netherlands)

VANDERKUIL, JHF; KORF, J

A method to monitor extracellular glucose in freely moving rats, based on intracerebral microdialysis coupled to an enzyme reactor is described. The dialysate is continuously mixed with a solution containing the enzymes hexokinase and glucose-6-phosphate dehydrogenase, and the fluorescence of NADPH

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

DEFF Research Database (Denmark)

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

2006-01-01

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

Contribution of PPARγ in modulation of acrolein-induced inflammatory signaling in gp91phox knock-out mice.

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Yousefipour, Zivar; Chug, Neha; Marek, Katarzyna; Nesbary, Alicia; Mathew, Joseph; Ranganna, Kasturi; Newaz, Mohammad A

2017-08-01

Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91 phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass) -1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass) -1 ·day -1 , orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

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Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap; Lee, Soh-Hyun; Ryoo, Zae Young; Chang, Kyu-Tae; Park, Jeen-Woo; Park, Dong-Chan; Song, Byoung J; Veech, Richard L; Song, Hebok; Huh, Tae-Lin

2004-09-17

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

Science.gov (United States)

Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

2015-08-01

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

Ghrelin receptor antagonism of hyperlocomotion in cocaine-sensitized mice requires βarrestin-2.

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Toth, Krisztian; Slosky, Lauren M; Pack, Thomas F; Urs, Nikhil M; Boone, Peter; Mao, Lan; Abraham, Dennis; Caron, Marc G; Barak, Lawrence S

2018-01-01

The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through βarrestin-2 to regulate actin/stress fiber rearrangement, suggesting βarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific βarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The βarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for βarrestin-2 and show that pharmacological inhibition of βarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of βarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate βarrestin-2 activity differentially from G protein activity may be required. © 2017 Wiley Periodicals, Inc.

Myeloid-specific deletion of NOX2 prevents the metabolic and neurologic consequences of high fat diet.

Directory of Open Access Journals (Sweden)

Jennifer K Pepping

Full Text Available High fat diet-induced obesity is associated with inflammatory and oxidative signaling in macrophages that likely participates in metabolic and physiologic impairment. One key factor that could drive pathologic changes in macrophages is the pro-inflammatory, pro-oxidant enzyme NADPH oxidase. However, NADPH oxidase is a pleiotropic enzyme with both pathologic and physiologic functions, ruling out indiscriminant NADPH oxidase inhibition as a viable therapy. To determine if targeted inhibition of monocyte/macrophage NADPH oxidase could mitigate obesity pathology, we generated mice that lack the NADPH oxidase catalytic subunit NOX2 in myeloid lineage cells. C57Bl/6 control (NOX2-FL and myeloid-deficient NOX2 (mNOX2-KO mice were given high fat diet for 16 weeks, and subject to comprehensive metabolic, behavioral, and biochemical analyses. Data show that mNOX2-KO mice had lower body weight, delayed adiposity, attenuated visceral inflammation, and decreased macrophage infiltration and cell injury in visceral adipose relative to control NOX2-FL mice. Moreover, the effects of high fat diet on glucose regulation and circulating lipids were attenuated in mNOX2-KO mice. Finally, memory was impaired and markers of brain injury increased in NOX2-FL, but not mNOX2-KO mice. Collectively, these data indicate that NOX2 signaling in macrophages participates in the pathogenesis of obesity, and reinforce a key role for macrophage inflammation in diet-induced metabolic and neurologic decline. Development of macrophage/immune-specific NOX-based therapies could thus potentially be used to preserve metabolic and neurologic function in the context of obesity.

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

Science.gov (United States)

Singh, Santosh; Trigun, Surendra K

2010-09-01

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

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

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

2016-01-01

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

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

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

2013-01-01

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

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

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Elaine Hatanaka

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

Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice.

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Miki Nagase

Full Text Available Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC and knockout (M-Rac1 KO mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by

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

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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.

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

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

2017-04-01

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

Retinoic acid activates two pathways required for meiosis in mice.

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Jana Koubova

2014-08-01

Full Text Available In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA, the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

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

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

2010-01-01

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

Use of NAD(P)H fluorescence measurement for on-line monitoring of metabolic state of Azohydromonas australica in poly(3-hydroxybutyrate) production.

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Gahlawat, Geeta; Srivastava, Ashok K

2013-02-01

Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3-12 h) and late log phase (24-39 h) of PHB fermentation. After 12 h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39 h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.

Crystal Structure of Perakine Reductase, Founding Member of a Novel Aldo-Keto Reductase (AKR) Subfamily That Undergoes Unique Conformational Changes during NADPH Binding*

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Sun, Lianli; Chen, Yixin; Rajendran, Chitra; Mueller, Uwe; Panjikar, Santosh; Wang, Meitian; Mindnich, Rebekka; Rosenthal, Cindy; Penning, Trevor M.; Stöckigt, Joachim

2012-01-01

Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His6-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His6-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α8/β6 barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family. PMID:22334702

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

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

2018-05-26

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

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

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

2010-01-01

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H)

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

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Sytykiewicz, Hubert

2016-07-22

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

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

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Efthimios A. Andronis

2014-04-01

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

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

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Bing-Hu Li

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

NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

DEFF Research Database (Denmark)

Panagiotou, Gianni; Christakopoulos, P.

2004-01-01

for NADPH over NADH. In this study, the influence of aeration and the response to the addition of electron acceptors on xylose fermentation by F. oxysporum were also studied. The batch cultivation of F. oxysporum on xylose was performed under aerobic, anaerobic and oxygen-limited conditions in stirred tank...... conditions (0.3 vvm). When the artificial electron acceptor acetoin was added to an anaerobic batch fermentation of xylose by F. oxysporum, the ethanol yield increased while xylitol excretion was also decreased....

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

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

2015-02-15

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

Live attenuated S. Typhimurium vaccine with improved safety in immuno-compromised mice.

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Balamurugan Periaswamy

Full Text Available Live attenuated vaccines are of great value for preventing infectious diseases. They represent a delicate compromise between sufficient colonization-mediated adaptive immunity and minimizing the risk for infection by the vaccine strain itself. Immune defects can predispose to vaccine strain infections. It has remained unclear whether vaccine safety could be improved via mutations attenuating a vaccine in immune-deficient individuals without compromising the vaccine's performance in the normal host. We have addressed this hypothesis using a mouse model for Salmonella diarrhea and a live attenuated Salmonella Typhimurium strain (ssaV. Vaccination with this strain elicited protective immunity in wild type mice, but a fatal systemic infection in immune-deficient cybb(-/-nos2(-/- animals lacking NADPH oxidase and inducible NO synthase. In cybb(-/-nos2(-/- mice, we analyzed the attenuation of 35 ssaV strains carrying one additional mutation each. One strain, Z234 (ssaV SL1344_3093, was >1000-fold attenuated in cybb(-/-nos2(-/- mice and ≈100 fold attenuated in tnfr1(-/- animals. However, in wt mice, Z234 was as efficient as ssaV with respect to host colonization and the elicitation of a protective, O-antigen specific mucosal secretory IgA (sIgA response. These data suggest that it is possible to engineer live attenuated vaccines which are specifically attenuated in immuno-compromised hosts. This might help to improve vaccine safety.

Dendrobium nobile Lindl. alkaloids regulate metabolism gene expression in livers of mice.

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Xu, Yun-Yan; Xu, Ya-Sha; Wang, Yuan; Wu, Qin; Lu, Yuan-Fu; Liu, Jie; Shi, Jing-Shan

2017-10-01

In our previous studies, Dendrobium nobile Lindl. alkaloids (DNLA) has been shown to have glucose-lowering and antihyperlipidaemia effects in diabetic rats, in rats fed with high-fat diets, and in mice challenged with adrenaline. This study aimed to examine the effects of DNLA on the expression of glucose and lipid metabolism genes in livers of mice. Mice were given DNLA at doses of 10-80 mg/kg, po for 8 days, and livers were removed for total RNA and protein isolation to perform real-time RT-PCR and Western blot analysis. Dendrobium nobile Lindl. alkaloids increased PGC1α at mRNA and protein levels and increased glucose metabolism gene Glut2 and FoxO1 expression. DNLA also increased the expression of fatty acid β-oxidation genes Acox1 and Cpt1a. The lipid synthesis regulator Srebp1 (sterol regulatory element-binding protein-1) was decreased, while the lipolysis gene ATGL was increased. Interestingly, DNLA increased the expression of antioxidant gene metallothionein-1 and NADPH quinone oxidoreductase-1 (Nqo1) in livers of mice. Western blot on selected proteins confirmed these changes including the increased expression of GLUT4 and PPARα. DNLA has beneficial effects on liver glucose and lipid metabolism gene expressions, and enhances the Nrf2-antioxidant pathway gene expressions, which could play integrated roles in regulating metabolic disorders. © 2017 Royal Pharmaceutical Society.

Effects of Differing Response-Force Requirements on Food-Maintained Responding in C57BL/6J Mice

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Zarcone, Troy J.; Chen, Rong; Fowler, Stephen C.

2009-01-01

The effect of force requirements on response effort was examined using inbred C57BL/6J mice trained to press a disk with their snout. Lateral peak forces greater than 2 g were defined as responses (i.e., all responses above the measurement threshold). Different, higher force requirements were used to define criterion responses (a subclass of all…

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

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

2009-09-15

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

Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic ¤Nicotiana sylvestris¤

DEFF Research Database (Denmark)

Michalecka, A.M.; Agius, S.C.; Møller, I.M.

2004-01-01

The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD...

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

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Partata W.A.

1999-01-01

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

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

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

1999-04-01

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

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

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Jadhao, Arun G; Biswas, Saikat P; Bhoyar, Rahul C; Pinelli, Claudia

2017-04-01

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential requirement for satellite cells during overload-induced muscle hypertrophy in growing versus mature mice.

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Murach, Kevin A; White, Sarah H; Wen, Yuan; Ho, Angel; Dupont-Versteegden, Esther E; McCarthy, John J; Peterson, Charlotte A

2017-07-10

Pax7+ satellite cells are required for skeletal muscle fiber growth during post-natal development in mice. Satellite cell-mediated myonuclear accretion also appears to persist into early adulthood. Given the important role of satellite cells during muscle development, we hypothesized that the necessity of satellite cells for adaptation to an imposed hypertrophic stimulus depends on maturational age. Pax7 CreER -R26R DTA mice were treated for 5 days with vehicle (satellite cell-replete, SC+) or tamoxifen (satellite cell-depleted, SC-) at 2 months (young) and 4 months (mature) of age. Following a 2-week washout, mice were subjected to sham surgery or 10 day synergist ablation overload of the plantaris (n = 6-9 per group). The surgical approach minimized regeneration, de novo fiber formation, and fiber splitting while promoting muscle fiber growth. Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area (CSA) were evaluated via immunohistochemistry. Myonuclei (myonuclei/100 mm) were counted on isolated single muscle fibers. Tamoxifen treatment depleted satellite cells by ≥90% and prevented myonuclear accretion with overload in young and mature mice (p overload. Average muscle fiber CSA increased ~20% in young SC+ (p = 0.07), mature SC+ (p overload (p overload-induced hypertrophy is dependent on maturational age, and global responses to overload differ in young versus mature mice.

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

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Morre, D. James

2002-01-01

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

Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

DEFF Research Database (Denmark)

Nissen, T.L.; Anderlund, M.; Nielsen, Jens

2001-01-01

The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/ADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can...... cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein...

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

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Perner, A; Andresen, Lars; Pedersen, G

2003-01-01

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

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

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

2014-07-10

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

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

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

2014-03-02

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

Effect of X-rays and u.v.-light on the levels of NAD(P), NAD(P)H and hydroxyproline in Pinus silvestris pollen

International Nuclear Information System (INIS)

Zelles, L.

1978-01-01

Pollen grains of Pinus Silvestris were irradiated with stimulating and inhibiting doses of X-rays and u.v.-light and the levels of NAD(P), NAD(P)H and hydroxyproline determined during tube growth. Pollen grains irradiated with stimulating doses of X-rays and u.v.-light developed longer tubes, while grains irradiated with inhibiting doses of X-rays and u.v.-light developed shorter tubes than the unirradiated controls. After 32 hr of incubation, the levels of NAD(P), NAD(P)H and hydroxyproline were at their lowest compared with unirradiated pollen. In samples with stimulating doses of irradiation NAD(P) reached its maximum earlier than in samples with inhibiting irradiation. The ratio between the concentrations of NAD(P) and NAD(P)H in the irradiated samples was higher than in the unirradiated control. The hydroxyproline content was higher in irradiated than in unirradiated pollen. (author)

The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae.

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Sacomboio, Euclides Nenga Manuel; Kim, Edson Yu Sin; Correa, Henrique Leonardo Ruchaud; Bonato, Paloma; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Chubatsu, Leda Satie; Müller-Santos, Marcelo

2017-10-19

The NTR system is the major regulator of nitrogen metabolism in Bacteria. Despite its broad and well-known role in the assimilation, biosynthesis and recycling of nitrogenous molecules, little is known about its role in carbon metabolism. In this work, we present a new facet of the NTR system in the control of NADPH concentration and the biosynthesis of molecules dependent on reduced coenzyme in Herbaspirillum seropedicae SmR1. We demonstrated that a ntrC mutant strain accumulated high levels of polyhydroxybutyrate (PHB), reaching levels up to 2-fold higher than the parental strain. In the absence of NtrC, the activity of glucose-6-phosphate dehydrogenase (encoded by zwf) increased by 2.8-fold, consequently leading to a 2.1-fold increase in the NADPH/NADP + ratio. A GFP fusion showed that expression of zwf is likewise controlled by NtrC. The increase in NADPH availability stimulated the production of polyhydroxybutyrate regardless the C/N ratio in the medium. The mutant ntrC was more resistant to H 2 O 2 exposure and controlled the propagation of ROS when facing the oxidative condition, a phenotype associated with the increase in PHB content.

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

Science.gov (United States)

Jakubowska, Dagmara; Janicka, Małgorzata

2017-11-01

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

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

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Jun Chen

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

Endogenous interleukin-12 is not required for resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection in mice.

Science.gov (United States)

Del Río, L; Buendía, A J; Sánchez, J; Gallego, M C; Caro, M R; Ortega, N; Seva, J; Pallarés, F J; Cuello, F; Salinas, J

2001-08-01

A Th1 immune response involving gamma interferon (IFN-gamma) production is required to eliminate Chlamydophila abortus infections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12(-/-) and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12(-/-) mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12(-/-) mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-gamma. The low level of IFN-gamma was essential for survival of IL-12(-/-) infected mice. Both WT and IL-12(-/-) mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-gamma and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge with C. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12(-/-) mice. The lack of IL-12 resulted in few infiltrating CD4(+) T cells in the liver relative to the number in WT mice, although the number of CD8(+) T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

International Nuclear Information System (INIS)

Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

2009-01-01

A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

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

Science.gov (United States)

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

2011-08-01

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

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

Science.gov (United States)

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

2013-12-13

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

PPARα Is Required for PPARδ Action in Regulation of Body Weight and Hepatic Steatosis in Mice

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Wojciech G. Garbacz

2015-01-01

Full Text Available Peroxisome proliferator activated receptors alpha (PPARα and delta (PPARδ belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent. Mice conditionally expressing human PPARδ demonstrated pronounced weight loss and promoted hepatic steatosis when treated with GW501516 (PPARδ-agonist when compared to wild type mice. This effect was completely absent in mice with either a dominant negative form of PPARδ or deletion of the DNA binding domain of PPARδ. This confirmed the absolute requirement for PPARδ in the physiological actions of GW501516 and confirmed the potential utility against the human form of this receptor. Surprisingly the genetic deletion of PPARα also abrogated the effect of GW501516 in terms of both weight loss and hepatic lipid accumulation. Also the levels of the PPARα endogenous agonist 16:0/18:1-GPC were shown to be modulated by PPARδ in wild type mice. Our results show that both PPARδ and PPARα receptors are essential for GW501516-driven adipose tissue reduction and subsequently hepatic steatosis, with PPARα working downstream of PPARδ.

Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

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Yuno Lee

Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

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Roy R Chaudhuri

2009-07-01

Full Text Available Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH, has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input with equivalent data produced after passage of the library through mice (output enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

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

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Lishan Zhou

2013-01-01

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

La p67-PHOX como un elemento esencial del sistema NADPH oxidasa de las células fagocíticas

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Pablo Javier Patiño Grajales

1999-01-01

Full Text Available Las células fagocíticas cumplen un papel fundamental en la defensa innata del huésped contra la invasión de microorganismos. Luego de ser fagocitados, uno de lo~ mecanismos utilizados para destruir dichos microorganismos es la producción de metabolitos intermedios del oxígeno generados a través del sistema NADPH oxidasa. Los radicales libres del oxígeno están al mismo tiempo involucrados en el daño tisular, que ocurre en muchas condiciones inflamatorias. La NADPH oxidasa es un complejo proteico conformado por varios componentes: gp91-phox y p22-phox, los cuales forman un heterodímero llamado citocromo b5581 que se encuentra en la membrana de las células fagocíticas, y por las proteínas p47-phox, p67-phox y p40-phox que forman un complejo macromolecular localizado en el citoplasma. Durante la activación de las células fagocíticas los componentes citosólicos del sistema se translocan a la membrana y se unen al citocromo b558' conformando así el complejo enzimático activo de la NADPH oxidasa cuya importancia biológica se ha evidenciado en las últimas cuatro décadas gracias al estudio de la enfermedad granulomatosa crónica (EGC. Ésta es una inmunodeficiencia primaria caracterizada por infecciones recurrentes y severas. Todos los casos de EGC reportados hasta ahora tienen que ver con una alteración en los genes que codifican las proteínas gp91-phox, p22-phox, p67-phoxy p47-phox. La p67 -phox es uno de los componentes del sistema NADPH oxidasa que más ha llamado la atención en los últimos años, pues tiene un papel fundamental en la activación del flujo de electrones desde el citoplasma a la vacuola fagocítica. En este articulo se hace una revisión acerca de lo que hasta ahora se conoce de la estructura y función de la p67-phox. Además se revisarán las diversas mutaciones identificadas en su gen, que debido a su alto grado de heterogeneidad son de gran utilidad para el entendimiento de la estructura y función del

Geraniol improves endothelial function by inhibiting NOX-2 derived oxidative stress in high fat diet fed mice

International Nuclear Information System (INIS)

Wang, Xiaoyu; Zhao, Shiqi; Su, Mengqi; Sun, Li; Zhang, Song; Wang, Dingyu; Liu, Zhaorui; Yuan, Yue; Liu, Yang; Li, Yue

2016-01-01

Endothelial dysfunction occurs in obese patients and high-fat diet (HFD) fed experimental animals. While geraniol has been reported to ameliorate inflammation and oxidative stress, inhibit tumor cell proliferation, and improve atherosclerosis, its direct effect on endothelial function remains uncharacterized. The present study therefore investigated the effect of geraniol on endothelial function in HFD mice and its underlying mechanisms. C57 BL/6 mice were fed an HFD (n = 40) or a normal diet (n = 20) for 8 weeks. HFD fed mice then were randomized to intraperitoneal treatment with geraniol (n = 20) or vehicle (n = 20) for another 6 weeks. Acetylcholine (Ach)-induced endothelial dependent vasorelaxation was measured on wire myography; reactive oxygen species (ROS) generation was assessed by fluorescence imaging, and NADPH oxidases (NOXs) and adhesive molecules VCAM-1 and ICAM-1 protein expression by western blotting. Geraniol improved endothelial function in HFD fed mice, as evidenced by its: 1. restoring endothelial dependent vasorelaxation induced by Ach, and reversing increased VCAM-1 and ICAM-1 expression; 2. attenuating HFD induced increased serum TBARS and aortic ROS generation; and 3. downregulating aortic NOX-2 expression in both HFD fed mice and in palmitic acid treated endothelial cells. Geraniol therefore protects against endothelial dysfunction induced by HFD through reducing NOX-2 associated ROS generation. -- Highlights: •Geraniol improved endothelial dependent relaxation in high fat diet fed mice. •Geraniol alleviated vascular injury in high fat diet fed mice. •Geraniol inhibited ROS generation through downregulating NOX-2 expression.

Geraniol improves endothelial function by inhibiting NOX-2 derived oxidative stress in high fat diet fed mice

Energy Technology Data Exchange (ETDEWEB)

Wang, Xiaoyu; Zhao, Shiqi; Su, Mengqi; Sun, Li; Zhang, Song; Wang, Dingyu; Liu, Zhaorui; Yuan, Yue; Liu, Yang [Department of Cardiology, the First Affiliated Hospital, Harbin Medical University, Harbin 150001, Heilongjiang Province (China); Li, Yue, E-mail: ly99ly@vip.163.com [Department of Cardiology, the First Affiliated Hospital, Harbin Medical University, Harbin 150001, Heilongjiang Province (China); Key Laboratory of Cardiac Diseases and Heart Failure, Harbin Medical University, Harbin, 150001, Heilongjiang Province (China)

2016-05-20

Endothelial dysfunction occurs in obese patients and high-fat diet (HFD) fed experimental animals. While geraniol has been reported to ameliorate inflammation and oxidative stress, inhibit tumor cell proliferation, and improve atherosclerosis, its direct effect on endothelial function remains uncharacterized. The present study therefore investigated the effect of geraniol on endothelial function in HFD mice and its underlying mechanisms. C57 BL/6 mice were fed an HFD (n = 40) or a normal diet (n = 20) for 8 weeks. HFD fed mice then were randomized to intraperitoneal treatment with geraniol (n = 20) or vehicle (n = 20) for another 6 weeks. Acetylcholine (Ach)-induced endothelial dependent vasorelaxation was measured on wire myography; reactive oxygen species (ROS) generation was assessed by fluorescence imaging, and NADPH oxidases (NOXs) and adhesive molecules VCAM-1 and ICAM-1 protein expression by western blotting. Geraniol improved endothelial function in HFD fed mice, as evidenced by its: 1. restoring endothelial dependent vasorelaxation induced by Ach, and reversing increased VCAM-1 and ICAM-1 expression; 2. attenuating HFD induced increased serum TBARS and aortic ROS generation; and 3. downregulating aortic NOX-2 expression in both HFD fed mice and in palmitic acid treated endothelial cells. Geraniol therefore protects against endothelial dysfunction induced by HFD through reducing NOX-2 associated ROS generation. -- Highlights: •Geraniol improved endothelial dependent relaxation in high fat diet fed mice. •Geraniol alleviated vascular injury in high fat diet fed mice. •Geraniol inhibited ROS generation through downregulating NOX-2 expression.

Increased sensitivity of apolipoprotein E knockout mice to copper-induced oxidative injury to the liver.

Science.gov (United States)

Chen, Yuan; Li, Bin; Zhao, Ran-ran; Zhang, Hui-feng; Zhen, Chao; Guo, Li

2015-04-10

Apolipoprotein E (ApoE) genotypes are related to clinical presentations in patients with Wilson's disease, indicating that ApoE may play an important role in the disease. However, our understanding of the role of ApoE in Wilson's disease is limited. High copper concentration in Wilson's disease induces excessive generation of free oxygen radicals. Meanwhile, ApoE proteins possess antioxidant effects. We therefore determined whether copper-induced oxidative damage differ in the liver of wild-type and ApoE knockout (ApoE(-/-)) mice. Both wild-type and ApoE(-/-) mice were intragastrically administered with 0.2 mL of copper sulfate pentahydrate (200 mg/kg; a total dose of 4 mg/d) or the same volume of saline daily for 12 weeks, respectively. Copper and oxidative stress markers in the liver tissue and in the serum were assessed. Our results showed that, compared with the wild-type mice administered with copper, TBARS as a marker of lipid peroxidation, the expression of oxygenase-1 (HO-1), NAD(P)H dehydrogenase, and quinone 1 (NQO1) significantly increased in the ApoE(-/-) mice administered with copper, meanwhile superoxide dismutase (SOD) activity significantly decreased. Thus, it is concluded that ApoE may protect the liver from copper-induced oxidative damage in Wilson's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2014-07-01

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

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Science.gov (United States)

Semprun-Prieto, Laura C.; Sukhanov, Sergiy; Yoshida, Tadashi; Rezk, Bashir M.; Gonzalez-Villalobos, Romer A.; Vaughn, Charlotte; Tabony, A. Michael; Delafontaine, Patrice

2011-01-01

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4 fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS. PMID:21570954

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

Science.gov (United States)

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

2009-09-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-08-15

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

Lycopene inhibits reactive oxygen species production in SK-Hep-1 cells and attenuates acetaminophen-induced liver injury in C57BL/6 mice.

Science.gov (United States)

Bandeira, Ana Carla Balthar; da Silva, Talita Prato; de Araujo, Glaucy Rodrigues; Araujo, Carolina Morais; da Silva, Rafaella Cecília; Lima, Wanderson Geraldo; Bezerra, Frank Silva; Costa, Daniela Caldeira

2017-02-01

Our aim was to investigate the antioxidant potential of lycopene in different experimental liver models: in vitro, to evaluate the influence of lycopene on reactive oxygen species (ROS) production mediated by the PKC pathway and in vivo, to evaluate the protective effects of lycopene in an experimental model of hepatotoxicity. The in vitro study assessed the lycopene antioxidant potential by the quantification of ROS production in SK-Hep-1 cells unstimulated or stimulated by an activator of the PKC pathway. The role of NADPH oxidase was evaluated by measuring its inhibition potential using an inhibitor of this enzyme. In the in vivo study, male C57BL/6 mice received lycopene (10 or 100 mg/kg by oral gavage) and 1 h later, acetaminophen (APAP) (500 mg/kg) was administrated. Lycopene decreased ROS production in SK-Hep-1 cells through inhibition of NADPH oxidase, brought about in the PKC pathway. Lycopene improved hepatotoxicity acting as an antioxidant, reduced GSSG and regulated tGSH and CAT levels, reduced oxidative damage primarily by decreasing protein carbonylation, promoted the downregulation of MMP-2 and reduced areas of necrosis improving the general appearance of the lesion in C57BL/6 mice. Lycopene is a natural compound that was able to inhibit the production of ROS in vitro and mitigate the damage caused by APAP overdose in vivo. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

International Nuclear Information System (INIS)

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

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

Science.gov (United States)

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

2017-10-01

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

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

Science.gov (United States)

Majumdar, Arkajo; Kar, Rup Kumar

2018-07-01

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

Dietary broccoli mildly improves neuroinflammation in aged mice but does not reduce lipopolysaccharide-induced sickness behavior.

Science.gov (United States)

Townsend, Brigitte E; Chen, Yung-Ju; Jeffery, Elizabeth H; Johnson, Rodney W

2014-11-01

Aging is associated with oxidative stress and heightened inflammatory response to infection. Dietary interventions to reduce these changes are therefore desirable. Broccoli contains glucoraphanin, which is converted to sulforaphane (SFN) by plant myrosinase during cooking preparation or digestion. Sulforaphane increases antioxidant enzymes including NAD(P)H quinone oxidoreductase and heme oxygenase I and inhibits inflammatory cytokines. We hypothesized that dietary broccoli would support an antioxidant response in brain and periphery of aged mice and inhibit lipopolysaccharide (LPS)-induced inflammation and sickness. Young adult and aged mice were fed control or 10% broccoli diet for 28 days before an intraperitoneal LPS injection. Social interactions were assessed 2, 4, 8, and 24 hours after LPS, and mRNA was quantified in liver and brain at 24 hours. Dietary broccoli did not ameliorate LPS-induced decrease in social interactions in young or aged mice. Interleukin-1β (IL-1β) expression was unaffected by broccoli consumption but was induced by LPS in brain and liver of adult and aged mice. In addition, IL-1β was elevated in brain of aged mice without LPS. Broccoli consumption decreased age-elevated cytochrome b-245 β, an oxidative stress marker, and reduced glial activation markers in aged mice. Collectively, these data suggest that 10% broccoli diet provides a modest reduction in age-related oxidative stress and glial reactivity, but is insufficient to inhibit LPS-induced inflammation. Thus, it is likely that SFN would need to be provided in supplement form to control the inflammatory response to LPS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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

OpenAIRE

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

2017-01-01

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

Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments

DEFF Research Database (Denmark)

Santos, Maria Margarida M. dos; Vijayendran, Raghevendran; Kotter, P.

2004-01-01

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two...

The Proatherogenic Effect of Chronic Nitric Oxide Synthesis Inhibition in ApoE-Null Mice Is Dependent on the Presence of PPARα

Directory of Open Access Journals (Sweden)

Michal Vechoropoulos

2014-01-01

Full Text Available Inhibition of endothelial nitric oxide synthase (eNOS accelerates atherosclerosis in ApoE-null mice by impairing the balance between angiotensin II (AII and NO. Our previous data suggested a role for PPARα in the deleterious effect of the renin-angiotensin system (RAS. We tested the hypothesis that ApoE-null mice lacking PPARα (DKO mice would be resistant to the proatherogenic effect of NOS inhibition. DKO mice fed a Western diet were immune to the 23% worsening in aortic sinus plaque area seen in the ApoE-null animals under 12 weeks of NOS inhibition with a subpressor dose of L-NAME, P=0.002. This was accompanied by a doubling of reactive oxygen species (ROS- generating aortic NADPH oxidase activity (a target of AII, which paralleled Nox1 expression and by a 10-fold excess of the proatherogenic iNOS, P<0.01. L-NAME also caused a doubling of aortic renin and angiotensinogen mRNA level in the ApoE-null mice but not in the DKO, and it upregulated eNOS in the DKO mice only. These data suggest that, in the ApoE-null mouse, PPARα contributes to the proatherogenic effect of unopposed RAS/AII action induced by L-NAME, an effect which is associated with Nox1 and iNOS induction, and is independent of blood pressure and serum lipids.

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli▿

Science.gov (United States)

Miller, E. N.; Jarboe, L. R.; Yomano, L. P.; York, S. W.; Shanmugam, K. T.; Ingram, L. O.

2009-01-01

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low Km values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural. PMID:19429550

Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

2016-01-01

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

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

Directory of Open Access Journals (Sweden)

Yan Li

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

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

Science.gov (United States)

McCarty, Mark F

2015-04-15

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

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

Czech Academy of Sciences Publication Activity Database

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

2012-01-01

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

Mechanism of mercuric chloride resistance in microorganisms. II. NADPH-dependent reduction of mercuric chloride and vaporization of mercury from mercuric chloride by a multiple drug resistant strain of Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Komura, I; Funaba, T; Izaki, K

1971-01-01

The activity to vaporize a /sup 203/Hg compound from /sup 203/HgCl/sub 2/ was demonstrated in crude cell-free extracts of a strain of Escherichia coli W2252, which had acquired the multiple drug resistance. NADPH was essential for the vaporization, while NADH had only a slight stimulating effect and NADP/sup +/ had no effect. The oxidation of NADPH dependent on HgCl/sub 2/ was also demonstrated in the crude extracts, but the HgCl/sub 2/-dependent NADH oxidation could be demonstrated only when a partially purified enzyme preparation was used. The rate of NADH oxidation was much slower than that of NADPH oxidation. It was concluded that NADPH, and to a lesser extent NADH, act as electron donors for the enzymatic reduction of HgCl/sub 2/ and the vaporization occurs after this reduction. This reduction and subsequent vaporization seem to provide a mechanism of resistance to HgCl/sub 2/ in E. coli strains having the multiple drug resistance. 15 references, 4 figures, 4 tables.

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice.

Science.gov (United States)

Northrup, Daniel; Yagi, Ryoji; Cui, Kairong; Proctor, William R; Wang, Chaochen; Placek, Katarzyna; Pohl, Lance R; Wang, Rongfu; Ge, Kai; Zhu, Jinfang; Zhao, Keji

2017-01-01

Natural killer (NK)T cells and conventional T cells share phenotypic characteristic however they differ in transcription factor requirements and functional properties. The role of histone modifying enzymes in conventional T cell development has been extensively studied, little is known about the function of enzymes regulating histone methylation in NKT cells. We show that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected. Loss of NKT cells is cell intrinsic and not due to an insufficient selection environment. The absence of NKT cells in UTX/JMJD3-deficient mice protects mice from concanavalin A-induced liver injury, a model of NKT-mediated hepatitis. GO-analysis of RNA-seq data indicates that cell cycle genes are downregulated in UTX/JMJD3-deleted NKT progenitors, and suggest that failed expansion may account for some of the cellular deficiency. The phenotype appears to be demethylase-dependent, because UTY, a homolog of UTX that lacks catalytic function, is not sufficient to restore their development and removal of H3K27me3 by deletion of EZH2 partially rescues the defect. NKT cell development and gene expression is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

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

NARCIS (Netherlands)

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

2010-01-01

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

IDH2 Deficiency Aggravates Fructose-Induced NAFLD by Modulating Hepatic Fatty Acid Metabolism and Activating Inflammatory Signaling in Female Mice

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Jeong Hoon Pan

2018-05-01

Full Text Available Fructose is a strong risk factor for non-alcoholic fatty liver disease (NAFLD, resulting from the disruption of redox systems by excessive reactive oxygen species production in the liver cells. Of note, recent epidemiological studies indicated that women are more prone to developing metabolic syndrome in response to fructose-sweetened beverages. Hence, we examined whether disruption of the redox system through a deletion of NADPH supplying mitochondrial enzyme, NADP+-dependent isocitrate dehydrogenase (IDH2, exacerbates fructose-induced NAFLD conditions in C57BL/6 female mice. Wild-type (WT and IDH2 knockout (KO mice were treated with either water or 34% fructose water over six weeks. NAFLD phenotypes and key proteins and mRNAs involved in the inflammatory pathway (e.g., NF-κB p65 and IL-1β were assessed. Hepatic lipid accumulation was significantly increased in IDH2 KO mice fed fructose compared to the WT counterpart. Neutrophil infiltration was observed only in IDH2 KO mice fed fructose. Furthermore, phosphorylation of NF-κB p65 and expression of IL-1β was remarkably upregulated in IDH2 KO mice fed fructose, and expression of IκBα was decreased by fructose treatment in both WT and IDH2 KO groups. For the first time, we report our novel findings that IDH2 KO female mice may be more susceptible to fructose-induced NAFLD and the associated inflammatory response, suggesting a mechanistic role of IDH2 in metabolic diseases.

Apocynin: Chemical and Biophysical Properties of a NADPH Oxidase Inhibitor

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Valdecir F. Ximenes

2013-03-01

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

Induction of fibroblast growth factor 21 does not require activation of the hepatic X-box binding protein 1 in mice

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Shantel Olivares

2017-12-01

Full Text Available Objective: Fibroblast growth factor 21 (FGF21, a key regulator of the metabolic response to fasting, is highly induced by endoplasmic reticulum (ER stress. The X-box binding protein 1 (Xbp1 is one of several ER stress proteins that has been shown to directly activate the FGF21 promoter. We aimed to determine whether hepatic Xbp1 is required for induction of hepatic FGF21 in vivo. Methods: Mice bearing a hepatocyte-specific deletion of Xbp1 (Xbp1LKO were subjected to fasting, pharmacologic ER stress, or a ketogenic diet, all potent stimuli of Fgf21 expression. Results: Hepatocyte-specific Xbp1 knockout mice demonstrated normal induction of FGF21 in response to fasting or pharmacologic ER stress and enhanced induction of FGF21 in response to a ketogenic diet. Consistent with preserved induction of FGF21, Xbp1LKO mice exhibited normal induction of FGF21 target genes and normal ketogenesis in response to fasting or a ketogenic diet. Conclusion: Hepatic Xbp1 is not required for induction of FGF21 under physiologic or pathophysiologic conditions in vivo. Keywords: Unfolded protein response, Endoplasmic reticulum stress, Fasting, Fatty acid oxidation, Ketogenic diet

Pulmonary oxidative stress is increased in cyclooxygenase-2 knockdown mice with mild pulmonary hypertension induced by monocrotaline.

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Francesca Seta

Full Text Available The aim of this study was to examine the role of cyclooxygenase-2 (COX-2 and downstream signaling of prostanoids in the pathogenesis of pulmonary hypertension (PH using mice with genetically manipulated COX-2 expression. COX-2 knockdown (KD mice, characterized by 80-90% suppression of COX-2, and wild-type (WT control mice were treated weekly with monocrotaline (MCT over 10 weeks. Mice were examined for cardiac hypertrophy/function and right ventricular pressure. Lung histopathological analysis was performed and various assays were carried out to examine oxidative stress, as well as gene, protein, cytokine and prostanoid expression. We found that MCT increased right ventricular systolic and pulmonary arterial pressures in comparison to saline-treated mice, with no evidence of cardiac remodeling. Gene expression of endothelin receptor A and thromboxane synthesis, regulators of vasoconstriction, were increased in MCT-treated lungs. Bronchoalveolar lavage fluid and lung sections demonstrated mild inflammation and perivascular edema but activation of inflammatory cells was not predominant under the experimental conditions. Heme oxygenase-1 (HO-1 expression and indicators of oxidative stress in lungs were significantly increased, especially in COX-2 KD MCT-treated mice. Gene expression of NOX-4, but not NOX-2, two NADPH oxidase subunits crucial for superoxide generation, was induced by ∼4-fold in both groups of mice by MCT. Vasodilatory and anti-aggregatory prostacyclin was reduced by ∼85% only in MCT-treated COX-2 KD mice. This study suggests that increased oxidative stress-derived endothelial dysfunction, vasoconstriction and mild inflammation, exacerbated by the lack of COX-2, contribute to the pathogenesis of early stages of PH when mild hemodynamic changes are evident and not yet accompanied by vascular and cardiac remodeling.

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

Science.gov (United States)

Koopmann, Edda; Hahlbrock, Klaus

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

International Nuclear Information System (INIS)

Koopmann, E.; Hahlbrock, K.

1997-01-01

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H

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

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Mark F. McCarty

2015-04-01

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

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

N-3 PUFAs protect against aortic inflammation and oxidative stress in angiotensin II-infused apolipoprotein E-/- mice.

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Kathryn M Wales

Full Text Available Abdominal aortic aneurysm is associated with infiltration of inflammatory cells into the aortic wall. The inflammatory response is also evident in animal models, such as apolipoprotein E-deficient (ApoE-/- mice that have been infused with angiotensin II, prior to development of aortic aneurysm. Since omega-3 polyunsaturated fatty acids (n-3 PUFAs and their metabolites have anti-inflammatory and pro-resolving activity, we hypothesised that dietary supplementation with n-3 PUFAs would protect against inflammatory processes in this mouse model. Twenty C57 and 20 ApoE-/- 3-4 week old male mice were supplemented with a low (0.14%, n = 10/group or high (0.70%, n = 10/group n-3 PUFA diet for 8 weeks before 2-day infusion with 0.9% saline or angiotensin II (1000 ng/kg/min. Four ApoE-/- mice on the low n-3 PUFA diet and none of the ApoE-/- mice on the high n-3 PUFA diet showed morphological evidence of abdominal aortic dissection. The plasma concentration of the n-3 PUFA metabolite, resolvin D1 was higher in angiotensin II-infused ApoE-/- mice fed the high, compared to the low n-3 PUFA diet. The number of neutrophils and macrophages infiltrating the abdominal aorta was elevated in ApoE-/- mice on the low n-3 PUFA diet, and this was significantly attenuated in mice that were fed the high n-3 PUFA diet. Most neutrophils and macrophages were associated with dissected aortas. Immunoreactivity of the catalytic subunit of nicotinamide-adenine dinucleotide phosphate (NADPH oxidase, Nox2, and superoxide were elevated in ApoE-/- mice that were fed the low n-3 PUFA diet, and this was also significantly attenuated in mice that were fed the high n-3 PUFA diet. Together, the findings indicate that supplementation of ApoE-/- mice with a diet high in n-3 PUFA content protected the mice against pro-inflammatory and oxidative stress responses following short-term infusion with angiotensin II.

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

Czech Academy of Sciences Publication Activity Database

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

2007-01-01

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

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

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Bo Young Jeong

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

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

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Varun Chandrashekaran

2017-10-01

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

Expresión y actividad de posibles polimorfismos provenientes de individuos normales en la proteína de 67 kd del sistema NADPH oxidasa utilizando el sistema COSphox.

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Andrés Augusto Arias

2004-09-01

Full Text Available El sistema NADPH oxidasa de las células fagocíticas cumple una función importante durante la respuesta antimicrobiana del organismo. La activación de este sistema está precedida por la translocación de las proteínas citosólicas p67phox, p47phox y p40phox hacia la membrana para ponerse en contacto con el flavocitocromo b558, lo que induce la generación del anión superóxido, un precursor de agentes microbicidas oxidantes. El presente trabajo presenta un análisis funcional del sistema NADPH oxidasa basado en los hallazgos de polimorfismos encontrados en el gen de p67phox de individuos sanos. Para esto se generaron mutaciones en el cADN que codifica la p67phox y se expresaron en el sistema de células COSphox. Los datos obtenidos en el presente trabajo indican que los cambios Val166?Ile, Pro329?Ser y His389?Gln no generan alteraciones en el funcionamiento de la p67phox cuando su función se analizó en el sistema transgénico basado en células COS-7. Por lo tanto, estos polimorfismos no generan ningún riesgo genético de producir deficiencias en la activación del sistema NADPH oxidasa. Además, se demuestra que el modelo de células COSphox representa un nuevo sistema celular, fácilmente transfectable que permite estudiar la función del sistema NADPH oxidasa de las células fagocíticas y sus particularidades genéticas. Finalmente, los hallazgos con estos polimorfismos nos permiten avanzar en el conocimiento sobre los mecanismos moleculares involucrados en la activación del sistema NADPH oxidasa células fagocíticas.

γδ T cells are required for pulmonary IL-17A expression after ozone exposure in mice: role of TNFα.

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Joel A Mathews

Full Text Available Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24-72 h. We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ-/- to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ-/- mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A+ CD45+ cells in the lungs and these effects were abolished in TCRδ-/- mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ-/- versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A+ CD45+ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A+ γδ T cells. Il17a mRNA and IL-17A+ γδ T cells were also lower in obese Cpefat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A+ γδ T cells to the lung.

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

DEFF Research Database (Denmark)

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

2016-01-01

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

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

DEFF Research Database (Denmark)

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

2010-01-01

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

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

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Meghan A Jendrysik

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

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

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Keith D Rochfort

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

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

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Hua, Fang; Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G.

2009-01-01

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.

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

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Yong Wang

2013-01-01

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

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

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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.

Treatment with salvianolic acid B restores endothelial function in angiotensin II-induced hypertensive mice.

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Ling, Wei Chih; Liu, Jian; Lau, Chi Wai; Murugan, Dharmani Devi; Mustafa, Mohd Rais; Huang, Yu

2017-07-15

Salvianolic acid B (Sal B) is one of the most abundant phenolic acids derived from the root of Danshen with potent anti-oxidative properties. The present study examined the vasoprotective effect of Sal B in hypertensive mice induced by angiotensin II (Ang II). Sal B (25mg/kg/day) was administered via oral gavage for 11days to Ang II (1.2mg/kg/day)-infused C57BL/6J mice (8-10weeks old). The vascular reactivity (both endothelium-dependent relaxations and contractions) in mouse arteries was examined by wire myography. The production of reactive oxygen species (ROS), protein level and localization of angiotensin AT 1 receptors and the proteins involved in ROS formation were evaluated using dihydroethidium (DHE) fluorescence, lucigenin-enhanced chemiluminescence, immunohistochemistry and Western blotting, respectively. The changes of ROS generating proteins were also assessed in vitro in human umbilical vein endothelial cells (HUVECs) exposed to Ang II with and without co-treatment with Sal B (0.1-10nM). Oral administration of Sal B reversed the Ang II-induced elevation of arterial systolic blood pressure in mice, augmented the impaired endothelium-dependent relaxations and attenuated the exaggerated endothelium-dependent contractions in both aortas and renal arteries of Ang II-infused mice. In addition, Sal B treatment normalized the elevated levels of AT 1 receptors, NADPH oxidase subunits (NOx-2 and NOx-4) and nitrotyrosine in arteries of Ang II-infused mice or in Ang II-treated HUVECs. In summary, the present study provided additional evidence demonstrating that Sal B treatment for 11days reverses the impaired endothelial function and with a marked inhibition of AT 1 receptor-dependent vascular oxidative stress. This vasoprotective and anti-oxidative action of Sal B most likely contributes to the anti-hypertensive action of the plant-derived compound. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

2012-01-01

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

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

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Tao-Cheng Wu

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

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

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

2007-02-20

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

The central cannabinoid CB1 receptor is required for diet-induced obesity and rimonabant's antiobesity effects in mice.

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Pang, Zhen; Wu, Nancy N; Zhao, Weiguang; Chain, David C; Schaffer, Erica; Zhang, Xin; Yamdagni, Preeti; Palejwala, Vaseem A; Fan, Chunpeng; Favara, Sarah G; Dressler, Holly M; Economides, Kyriakos D; Weinstock, Daniel; Cavallo, Jean S; Naimi, Souad; Galzin, Anne-Marie; Guillot, Etienne; Pruniaux, Marie-Pierre; Tocci, Michael J; Polites, H Greg

2011-10-01

Cannabinoid receptor CB1 is expressed abundantly in the brain and presumably in the peripheral tissues responsible for energy metabolism. It is unclear if the antiobesity effects of rimonabant, a CB1 antagonist, are mediated through the central or the peripheral CB1 receptors. To address this question, we generated transgenic mice with central nervous system (CNS)-specific knockdown (KD) of CB1, by expressing an artificial microRNA (AMIR) under the control of the neuronal Thy1.2 promoter. In the mutant mice, CB1 expression was reduced in the brain and spinal cord, whereas no change was observed in the superior cervical ganglia (SCG), sympathetic trunk, enteric nervous system, and pancreatic ganglia. In contrast to the neuronal tissues, CB1 was undetectable in the brown adipose tissue (BAT) or the liver. Consistent with the selective loss of central CB1, agonist-induced hypothermia was attenuated in the mutant mice, but the agonist-induced delay of gastrointestinal transit (GIT), a primarily peripheral nervous system-mediated effect, was not. Compared to wild-type (WT) littermates, the mutant mice displayed reduced body weight (BW), adiposity, and feeding efficiency, and when fed a high-fat diet (HFD), showed decreased plasma insulin, leptin, cholesterol, and triglyceride levels, and elevated adiponectin levels. Furthermore, the therapeutic effects of rimonabant on food intake (FI), BW, and serum parameters were markedly reduced and correlated with the degree of CB1 KD. Thus, KD of CB1 in the CNS recapitulates the metabolic phenotype of CB1 knockout (KO) mice and diminishes rimonabant's efficacy, indicating that blockade of central CB1 is required for rimonabant's antiobesity actions.

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

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

2018-05-01

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

JST Thesaurus Headwords and Synonyms: チオレドキシンレダクターゼ(NADPH) [MeCab user dictionary for science technology term[Archive

Lifescience Database Archive (English)

Full Text Available MeCab user dictionary for science technology term チオレドキシンレダクターゼ(NADPH) 名詞 一般 * * * ...* チオレドキシンレダクターゼ チオレドキシンレダクターゼ チオレドキシンレダクターゼ Thesaurus2015 200906077629637990 C LS38 UNKNOWN_2 チオレドキシンレダクターゼ ( NADPH )

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.

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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.

The Forkhead Transcription Factor, FOXP3, Is Required for Normal Pituitary Gonadotropin Expression in Mice1

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Jung, Deborah O.; Jasurda, Jake S.; Egashira, Noboru; Ellsworth, Buffy S.

2012-01-01

ABSTRACT The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3sf/Y). Although the gonadotroph cells appear to be intact in Foxp3sf/Y mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3sf/Y males. Treatment of Foxp3sf/Y males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3sf/Y males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3sf/Y males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

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Hua, Fang, E-mail: fhua2@emory.edu [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States); Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G. [Department of Emergency Medicine, Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Suite 5100, Atlanta, GA 30322 (United States)

2009-12-18

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-{kappa}B). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-{kappa}B and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-{kappa}B activity and phosphorylation of the inhibitor of kappa B (I{kappa}B{alpha}) increased in ischemic brains, but IRF3, inhibitor of {kappa}B kinase complex-{epsilon} (IKK{epsilon}), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-{kappa}B activity or p-I{kappa}B{alpha} induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-{kappa}B signaling and brain injury after acute cerebral I/R.

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases ▿ †

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Heer, Dominik; Heine, Daniel; Sauer, Uwe

2009-01-01

Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, 13C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPH-dependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype. PMID:19854918

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

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Simon Auer

2017-05-01

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

Hepatoprotective Effects of Ixora parviflora Extract against Exhaustive Exercise-Induced Oxidative Stress in Mice

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Chi-Chang Huang

2013-09-01

Full Text Available Ixora parviflora, a species of the Rubiaceae, is rich in polyphenols and flavonoids, and has been traditionally used as a folk medicine. An I. parviflora extract (IPE has great antioxidant activity in vitro, including a scavenging effect on superoxide radicals, reducing power, and ferrous ion-chelating ability. However, whether IPE is efficacious against oxidative damage in vivo is not known. The purpose of this study was to determine the protective effects of IPE treatment on hepatic oxidative stress and antioxidant defenses after exhaustive exercise in mice. Fifty male C57BL/6 mice (6 week old were randomly divided into five groups and designated a sedentary control with vehicle (C, and exhaustive exercise with vehicle (IPE0, low dosage (IPE10, medium dosage (IPE50 and high dosage (IPE100 of IPE at 0, 10, 50, and 100 mg/kg, respectively. After a single bout of exhaustive swimming exercise challenge, levels of blood ammonia and creatine kinase (CK, and hepatic superoxide dismutase (SOD protein expression, thiobarbituric acid-reactive substance (TBARS, and gp91phox, p22phox, and p47phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressions in the IPE0 group were significantly affected compared to those of the C group, but they were all significantly inhibited by the IPE treatments. Results of the present in vivo study in mice indicate that I. parviflora extract possesses antioxidative and hepatoprotective potential following exhaustive exercise.

GIMAP6 is required for T cell maintenance and efficient autophagy in mice.

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Pascall, John C; Webb, Louise M C; Eskelinen, Eeva-Liisa; Innocentin, Silvia; Attaf-Bouabdallah, Noudjoud; Butcher, Geoffrey W

2018-01-01

The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.

Decreased insulin secretory response of pancreatic islets during culture in the presence of low glucose is associated with diminished 45Ca2+ net uptake, NADPH/NADP+ and GSH/GSSG ratios

International Nuclear Information System (INIS)

Verspohl, E.J.; Kaiser, P.; Wahl, M.; Ammon, H.P.T.

1988-01-01

In isolated rat pancreatic islets maintained at a physiologic glucose concentration (5.6 mM) the effect of glucose on parameters which are known to be involved in the insulin secretion coupling such as NADPH, reduced glutathione (GSH), 86 Rb + efflux, and 45 Ca ++ net uptake were investigated. The insulinotropic effect of 16.7 mM glucose was decreased with the period of culturing during the first 14 days being significant after 2 days though in control experiments both protein content and ATP levels per islet were not affected and insulin content was only slightly decreased. Both NADPH and GSH decreased with time of culture. 86 Rb + efflux which is decreased by enhancing the glucose concentration from 3 to 5.6 mM in freshly isolated islets was not affected by culturing whatsoever, even not after 14 days of culture when there was not longer any insulin responsiveness to glucose. The 45 Ca ++ net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2) that more likely it is the result of a diminished 45 Ca ++ net uptake as a consequence of the inability of islet cells to maintain proper NADPH and GSH levels, and (3) that potassium ( 86 Rb + ) efflux may not be related to changes of NADPH and GSH

Redox imbalance due to the loss of mitochondrial NAD(P)-transhydrogenase markedly aggravates high fat diet-induced fatty liver disease in mice.

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Navarro, Claudia D C; Figueira, Tiago R; Francisco, Annelise; Dal'Bó, Genoefa A; Ronchi, Juliana A; Rovani, Juliana C; Escanhoela, Cecilia A F; Oliveira, Helena C F; Castilho, Roger F; Vercesi, Anibal E

2017-12-01

The mechanisms by which a high fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunctions and redox imbalance. We hypothesized that a HFD would increase mitochondrial reliance on NAD(P)-transhydrogenase (NNT) as the source of NADPH for antioxidant systems that counteract NAFLD development. Therefore, we studied HFD-induced liver mitochondrial dysfunctions and NAFLD in C57Unib.B6 congenic mice with (Nnt +/+ ) or without (Nnt -/- ) NNT activity; the spontaneously mutated allele (Nnt -/- ) was inherited from the C57BL/6J mouse substrain. After 20 weeks on a HFD, Nnt -/- mice exhibited a higher prevalence of steatohepatitis and content of liver triglycerides compared to Nnt +/+ mice on an identical diet. Under a HFD, the aggravated NAFLD phenotype in the Nnt -/- mice was accompanied by an increased H 2 O 2 release rate from mitochondria, decreased aconitase activity (a redox-sensitive mitochondrial enzyme) and higher susceptibility to Ca 2+ -induced mitochondrial permeability transition. In addition, HFD led to the phosphorylation (inhibition) of pyruvate dehydrogenase (PDH) and markedly reduced the ability of liver mitochondria to remove peroxide in Nnt -/- mice. Bypass or pharmacological reactivation of PDH by dichloroacetate restored the peroxide removal capability of mitochondria from Nnt -/- mice on a HFD. Noteworthy, compared to mice that were chow-fed, the HFD did not impair peroxide removal nor elicit redox imbalance in mitochondria from Nnt +/+ mice. Therefore, HFD interacted with Nnt mutation to generate PDH inhibition and further suppression of peroxide removal. We conclude that NNT plays a critical role in counteracting mitochondrial redox imbalance, PDH inhibition and advancement of NAFLD in mice fed a HFD. The present study provide seminal experimental evidence that redox imbalance in liver mitochondria potentiates the progression from simple steatosis to steatohepatitis following a HFD. Copyright

The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice

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Flávia Rezende

2018-05-01

Innovation and conclusion: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.

The Synergistic Effect of Valsartan and LAF237 [(S)-1-[(3-Hydroxy-1-Adamantyl)Ammo]acetyl-2-Cyanopyrrolidine] on Vascular Oxidative Stress and Inflammation in Type 2 Diabetic Mice

Science.gov (United States)

Shen, Min; Sun, Dongdong; Li, Weijie; Liu, Bing; Wang, Shenxu; Zhang, Zheng; Cao, Feng

2012-01-01

Aim. To investigate the combination effects and mechanisms of valsartan (angiotensin II type 1 receptor blocker) and LAF237 (DPP-IV inhibitor) on prevention against oxidative stress and inflammation injury in db/db mice aorta. Methods. Db/db mice (n = 40) were randomized to receive valsartan, LAF237, valsartan plus LAF237, or saline. Oxidative stress and inflammatory reaction in diabetic mice aorta were examined. Results. Valsartan or LAF237 pretreatment significantly increased plasma GLP-1 expression, reduced apoptosis of endothelial cells isolated from diabetic mice aorta. The expression of NAD(P)H oxidase subunits also significantly decreased resulting in decreased superoxide production and ICAM-1 (fold change: valsartan : 7.5 ± 0.7, P valsartan : 5.2 ± 1.2, P valsartan: 3.2 ± 0.6, LAF237: 4.7 ± 0.8; P valsartan and LAF237 resulted in a more significant increase of GLP-1 expression. The decrease of the vascular oxidative stress and inflammation reaction was also higher than monotherapy with valsartan or LAF237. Conclusion. These data indicated that combination treatment with LAF237 and valsartan acts in a synergistic manner on vascular oxidative stress and inflammation in type 2 diabetic mice. PMID:22474415

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

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Branka Marković

2017-01-01

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

Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy.

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Zhao, Man; Gao, Liang; Zhang, Li; Bai, Yanbin; Chen, Liang; Yu, Meilan; Cheng, Feng; Sun, Jie; Wang, Zhao; Ying, Xiangxian

2017-11-01

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL]. The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%. Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

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

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

2013-01-01

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

Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

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Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

2009-01-01

The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

Astragaloside IV Prevents Cardiac Remodeling in the Apolipoprotein E-Deficient Mice by Regulating Cardiac Homeostasis and Oxidative Stress

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Xiong-Zhi Li

2017-12-01

Full Text Available Background: Hypercholesterolemia is a risk factor for the development of cardiac hypertrophy. Astragaloside IV (AST-IV possesses cardiovascular protective properties. We hypothesize that AST-IV prevents cardiac remodeling with hypercholesterolemia via modulating tissue homeostasis and alleviating oxidative stress. Methods: The ApoE-/- mice were treated with AST-IV at 1 or 10 mg/kg for 8 weeks. The blood lipids tests, echocardiography, and TUNEL were performed. The mRNA expression profile was detected by real-time PCR. The myocytes size and number, and the expressions of proliferation (ki67, senescence (p16INK4a, oxidant (NADPH oxidase 4, NOX4 and antioxidant (superoxide dismutase, SOD were observed by immunofluorescence staining. Results: Neither 1 mg/kg nor 10 mg/kg AST-IV treatment could decrease blood lipids in ApoE-/- mice. However, the decreased left ventricular ejection fraction (LVEF and fractional shortening (FS in ApoE–/– mice were significantly improved after AST-IV treatment. The cardiac collagen volume fraction declined nearly in half after AST-IV treatment. The enlarged myocyte size was suppressed, and myocyte number was recovered, and the alterations of genes expressions linked to cell cycle, proliferation, senescence, p53-apoptosis pathway and oxidant-antioxidants in the hearts of ApoE-/- mice were reversed after AST-IV treatment. The decreased ki67 and increased p16INK4a in the hearts of ApoE-/- mice were recovered after AST-IV treatment. The percentages of apoptotic myocytes and NOX4-positive cells in AST-IV treated mice were decreased, which were consistent with the gene expressions. Conclusion: AST-IV treatment could prevent cardiac remodeling and recover the impaired ventricular function induced by hypercholesterolemia. The beneficial effect of AST-IV might partly be through regulating cardiac homeostasis and anti-oxidative stress.

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

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

2015-06-01

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

[Localization of NADPH-diaphorase in the brain of the shore crab Hemigrapsus sanguineus].

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Kotsiuba, E P

2005-01-01

The presence and localization of NADPH-diaphorase in the cerebral ganglion of the shore crab Hemigrapsus sanguineus was investigated with histochemical and electron histochemical methods. The reactivity of this enzyme was found in the deutrocerebrum, mainly in neuropils of olfactory lobes, the lateral antennular neuropil, a laterodorsal group of cells, and in the oculomotor nerve nucleus. Ultrastructural localization of the enzyme was detected in neurons on the perinuclear membrane, and in membranes of endoplasmic reticulum, in mitochondria and cytosol. The enzyme was found in axons of the antennular nerve, and in terminals of receptor axons in the glomerulus. The obtained data testify to participation of NO in perception and processing of the olfactory information.

Apocynin suppresses the progression of atherosclerosis in apoE-deficient mice by inactivation of macrophages

International Nuclear Information System (INIS)

Kinoshita, Hiroyuki; Matsumura, Takeshi; Ishii, Norio; Fukuda, Kazuki; Senokuchi, Takafumi; Motoshima, Hiroyuki; Kondo, Tatsuya; Taketa, Kayo; Kawasaki, Shuji; Hanatani, Satoko; Takeya, Motohiro; Nishikawa, Takeshi; Araki, Eiichi

2013-01-01

Highlights: ► We examined the anti-athrogenic effect of apocynin in atherosclerotic model mice. ► Apocynin prevented atherosclerotic lesion formation. ► Apocynin suppressed ROS production in aorta and in macrophages. ► Apocynin suppressed cytokine expression and cell proliferation in macrophages. ► Apocynin may be beneficial compound for the prevention of atherosclerosis. -- Abstract: Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages plays an important role in atherogenesis. Apocynin (4-hydroxy-3-methoxy-acetophenone), which is well known as a NADPH oxidase inhibitor, has anti-inflammatory effects including suppression of the generation of ROS. However, the suppressive effects of apocynin on the progression of atherosclerosis are not clearly understood. Thus, we investigated anti-atherosclerotic effects of apocynin using apolipoprotein E-deficient (apoE –/– ) mice in vivo and in mouse peritoneal macrophages in vitro. In atherosclerosis-prone apoE –/– mice, apocynin suppressed the progression of atherosclerosis, decreased 4-hydroxynonenal-positive area in atherosclerotic lesions, and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in aorta. In mouse peritoneal macrophages, apocynin suppressed the Ox-LDL-induced ROS generation, mRNA expression of MCP-1, IL-6 and granulocyte/macrophage colony-stimulating factor, and cell proliferation. Moreover, immunohistochemical studies revealed that apocynin decreased the number of proliferating cell nuclear antigen-positive macrophages in atherosclerotic lesions of apoE –/– mice. These results suggested that apocynin suppressed the formation of atherosclerotic lesions, at least in part, by inactivation of macrophages. Therefore, apocynin may be a potential therapeutic material to prevent the progression of atherosclerosis

Apocynin suppresses the progression of atherosclerosis in apoE-deficient mice by inactivation of macrophages

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Kinoshita, Hiroyuki [Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556 (Japan); Matsumura, Takeshi, E-mail: takeshim@gpo.kumamoto-u.ac.jp [Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556 (Japan); Ishii, Norio; Fukuda, Kazuki; Senokuchi, Takafumi; Motoshima, Hiroyuki; Kondo, Tatsuya; Taketa, Kayo; Kawasaki, Shuji; Hanatani, Satoko [Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556 (Japan); Takeya, Motohiro [Department of Cell Pathology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556 (Japan); Nishikawa, Takeshi; Araki, Eiichi [Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556 (Japan)

2013-02-08

Highlights: ► We examined the anti-athrogenic effect of apocynin in atherosclerotic model mice. ► Apocynin prevented atherosclerotic lesion formation. ► Apocynin suppressed ROS production in aorta and in macrophages. ► Apocynin suppressed cytokine expression and cell proliferation in macrophages. ► Apocynin may be beneficial compound for the prevention of atherosclerosis. -- Abstract: Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages plays an important role in atherogenesis. Apocynin (4-hydroxy-3-methoxy-acetophenone), which is well known as a NADPH oxidase inhibitor, has anti-inflammatory effects including suppression of the generation of ROS. However, the suppressive effects of apocynin on the progression of atherosclerosis are not clearly understood. Thus, we investigated anti-atherosclerotic effects of apocynin using apolipoprotein E-deficient (apoE{sup –/–}) mice in vivo and in mouse peritoneal macrophages in vitro. In atherosclerosis-prone apoE{sup –/–} mice, apocynin suppressed the progression of atherosclerosis, decreased 4-hydroxynonenal-positive area in atherosclerotic lesions, and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in aorta. In mouse peritoneal macrophages, apocynin suppressed the Ox-LDL-induced ROS generation, mRNA expression of MCP-1, IL-6 and granulocyte/macrophage colony-stimulating factor, and cell proliferation. Moreover, immunohistochemical studies revealed that apocynin decreased the number of proliferating cell nuclear antigen-positive macrophages in atherosclerotic lesions of apoE{sup –/–} mice. These results suggested that apocynin suppressed the formation of atherosclerotic lesions, at least in part, by inactivation of macrophages. Therefore, apocynin may be a potential therapeutic material to prevent the progression of atherosclerosis.

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

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

2017-06-01

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

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

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

2013-07-01

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

QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

Science.gov (United States)

Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.ABSTRACTDNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

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

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NEPOMUCENO ALEXANDRE LIMA

2000-01-01

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

Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

Science.gov (United States)

Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

2017-09-01

Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

Selection for high and low oxygen consumption-induced differences in maintenance energy requirements of mice.

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Darhan, Hongyu; Kikusato, Motoi; Toyomizu, Masaaki; Roh, Sang-Gun; Katoh, Kazuo; Sato, Masahiro; Suzuki, Keiichi

2017-07-01

Maintenance energy requirements (MER) of mice selected for high (H) or low (L) oxygen consumption (OC) were compared. Forty-four mice from H and L OC lines were weaned at 3 weeks and divided into four experimental groups: group A were sacrificed at 4 weeks; group B were fed ad libitum, and groups C and D were fed 2.8 and 2.4 g/day, respectively, from 4 to 8 weeks of age. Groups B-D were sacrificed at 8 weeks. Chemical components were estimated for all groups. MER was estimated using a model that partitioned metabolizable energy intake into that used for maintenance, and protein and fat deposition. The feed conversion ratio for the B group was significantly higher in the H than in the L line. Feed intake for metabolic energy content per metabolic body size was significantly also higher in the H line, whereas accumulated energy content per metabolic body size was significantly higher in the L line. MER of the H line was greater than that of the L line (P < 0.10). These results suggest that selection for H or L OC produced differences in chemical components, feed efficiency, and MER between the H and L lines. © 2016 Japanese Society of Animal Science.

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

Science.gov (United States)

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2017-12-05

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

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

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Lee I-Ta

2012-11-01

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

Glycosidic Bond Cleavage is Not Required for Phytosteryl Glycoside-Induced Reduction of Cholesterol Absorption in Mice

Science.gov (United States)

Lin, Xiaobo; Ma, Lina; Moreau, Robert A.

2012-01-01

Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with tracers cholesterol-d7 and sitostanol-d4. In a three-day fecal recovery study, ASG reduced cholesterol absorption efficiency by 45 ± 6% compared with 40 ± 6% observed with PSE. Four hours after gavage, plasma and liver cholesterol-d7 levels were reduced 86% or more when ASG was present. Liver total phytosterols were unchanged after ASG administration but were significantly increased after PSE. After ASG treatment both ASG and deacylated steryl glycosides (SG) were found in the gut mucosa and lumen. ASG was quantitatively recovered from stool samples as SG. These results demonstrate that ASG reduces cholesterol absorption in mice as efficiently as PSE while having little systemic absorption itself. Cleavage of the glycosidic linkage is not required for biological activity of ASG. Phytosteryl glycosides should be included in measurements of bioactive phytosterols. PMID:21538209

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

DEFF Research Database (Denmark)

Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt

2012-01-01

(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC......The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling...... with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP...

Correlation of denitrification-accepted fraction of electrons with NAD(P)H fluorescence for Pseudomonas aeruginosa performing simultaneous denitrification and respiration at extremely low dissolved oxygen conditions.

Science.gov (United States)

Chen, Fan; Xia, Qing; Ju, Lu-Kwang

2004-01-01

In cystic fibrosis airway infection, Pseudomonas aeruginosa forms a microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium's metabolism at microaerobic conditions. In this work, the culture properties and two indicators (the denitrification-accepted e- fraction and an NAD(P)H fluorescence fraction) for the culture's "fractional approach" to a fully anaerobic denitrifying state were examined in continuous cultures with practically zero DO but different aeration rates. With decreasing aeration, specific OUR decreased while specific NAR and NIR increased and kept Y(ATP/S) relatively constant. P. aeruginosa thus appeared to effectively compensate for energy generation at microaerobic conditions with denitrification. At the studied dilution rate of 0.06 h(-1), the maximum specific OUR was 2.8 mmol O2/g cells-h and the Monod constant for DO, in the presence of nitrate, was extremely low (Y(X/S) increased significantly (from 0.24 to 0.34) with increasing aeration, attributed to a roughly opposite trend of Y(ATP/X) (ATP generation required for cell growth). As for the denitrification-accepted e- fraction and the fluorescence fraction, both decreased with increasing aeration as expected. The two fractions, however, were not directly proportional. The fluorescence fraction changed more rapidly than the e- fraction at very low aeration rates, whereas the opposite was true at higher aeration. The results demonstrated the feasibility of using online NAD(P)H fluorescence to monitor sensitive changes of cellular physiology and provided insights to the shift of e- -accepting mechanisms of P. aeruginosa under microaerobic conditions.

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

Science.gov (United States)

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

2017-04-01

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

B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice.

Science.gov (United States)

Charlton, B; Zhang, M D; Slattery, R M

2001-12-01

Previous studies have implicated B lymphocytes in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse. While it is clear that B lymphocytes are necessary, it has not been clear at which stage of disease they play a role; early, late or both. To clarify when B lymphocytes are needed, T lymphocytes were transferred from 5-week-old NOD female mice to age-matched NOD/severe combined immunodeficiency (SCID) recipient mice. NOD/SCID mice, which lack functionally mature T and B lymphocytes, do not normally develop insulitis or insulin-dependent diabetes melitus (IDDM). The NOD/SCID mice that received purified T lymphocytes from 5-week-old NOD mice subsequently developed insulitis and diabetes even though they did not have detectable B lymphocytes. This suggests that while B lymphocytes may be essential for an initial priming event they are not requisite for disease progression in the NOD mouse.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

Energy Technology Data Exchange (ETDEWEB)

Maeda, Tomoji, E-mail: t-maeda@nichiyaku.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Nakajima, Toshihiro [Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku, Shinjyuku, Tokyo, Tokyo, 160-8402 (Japan); Komano, Hiroto, E-mail: hkomano@iwate-med.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan)

2016-05-13

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

International Nuclear Information System (INIS)

Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

2016-01-01

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

Efficient regeneration of NADPH in a 3-enzyme cascade reaction by in situ generation of glucose 6-phosphate from glucose and pyrophosphate

NARCIS (Netherlands)

Hartog, A.F.; van Herk, T.; Wever, R.

2011-01-01

We report here a promising method to regenerate NADPH (nicotinamide adenine dinucleotide phosphate) using the intermediate formation of glucose 6-phosphate (G6P) from glucose and pyrophosphate (PPi) catalyzed by the acid phosphatase from Shigella flexneri (PhoN-Sf). The G6P formed is used in turn by

Anti-diabetic effects of DA-11004, a synthetic IDPc inhibitor in high fat high sucrose diet-fed C57BL/6J mice.

Science.gov (United States)

Shin, Chang Yell; Jung, Mi Young; Lee, In Ki; Son, Miwon; Kim, Dong Sung; Lim, Joong In; Kim, Soon Hoe; Yoo, Moohi; Huh, Tae Lin; Sohn, Young Taek; Kim, Won Bae

2004-01-01

DA-11004 is a synthetic, potent NADP-dependent isocitrate dehydrogenase (IDPc) inhibitor where IC50 for IDPc is 1.49 microM. The purpose of this study was to evaluate the effects of DA-11004 on the high fat high sucrose (HF)-induced obesity in male C57BL/6J mice. After completing a 8-week period of experimentation, the mice were sacrificed 1 hr after the last DA-11004 treatment and their blood, liver, and adipose tissues (epididymal and retroperitoneal fat) were collected. There was a significant difference in the pattern of increasing body weight between the HF control and the DA-11004 group. In the DA-11004 (100 mg/kg) treated group the increase in body weight significantly declined and a content of epididymal fat and retroperitoneal fat was also significantly decreased as opposed to the HF control. DA-11004 (100 mg/ kg) inhibited the IDPc activity, and thus, NADPH levels in plasma and the levels of free fatty acid (FFA) or glucose in plasma were less than the levels of the HF control group. In conclusion, DA-11004 inhibited the fatty acid synthesis in adipose tissues via IDPc inhibition, and it decreased the plasma glucose levels and FFA in HF diet-induced obesity of C57BL/6J mice.

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

Directory of Open Access Journals (Sweden)

Wilson Belinda

2007-09-01

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

NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis

Energy Technology Data Exchange (ETDEWEB)

Timko, Michael P

2013-02-01

The biosynthesis of chlorophyll is a critical biochemical step in the development of photosynthetic vascular plants and green algae. From photosynthetic bacteria (cyanobacteria) to algae, non-vascular plants, gymnosperms and vascular plants, mechanisms have evolved for protochlorophyllide reduction a key step in chlorophyll synthesis. Protochlorophyllide reduction is carried out by both a light-dependent (POR) and light-independent (LIPOR) mechanisms. NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33, abbreviated POR) catalyzes the light-dependent reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide). In contrast, a light-independent protochlorophyllide reductase (LIPOR) involves three plastid gene products (chlL, chlN, and chlB) and several nuclear factors. Our work focused on characterization of both the POR and LIPOR catalyzed processes.

JST Thesaurus Headwords and Synonyms: NAD(P)H OXYDASE [MeCab user dictionary for science technology term[Archive

Lifescience Database Archive (English)

Full Text Available MeCab user dictionary for science technology term NAD(P)H OXYDASE 名詞 一般 * * * * ＮＡＤ...（Ｐ）Ｈオキシダーゼ ＮＡＤ（Ｐ）Ｈオキシダーゼ エヌエイディー（ピー）エイチオキシダーゼ Thesaurus2015 200906089709028550 C LS38 UNKNOWN_2 NAD ( P ) H OXYDASE

In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess apoE

NARCIS (Netherlands)

Dijk, K.W. van; Vlijmen, B.J.M. van; Hof, H.B. van 't; Zee, A. van der; Santamarina-Fojo, S.; Berkel, T.J.C. van; Havekes, L.M.; Hofker, M.H.

1999-01-01

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes

Rice Rab11 is required for JA-mediated defense signaling

Energy Technology Data Exchange (ETDEWEB)

Hong, Min Ji [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of); Lee, Yun mi [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Son, Young Sim [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Im, Chak Han [Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360 (Korea, Republic of); Yi, Young Byung [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Rim, Yeong Gil [Systems and Synthetic Agrobiotech Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Bahk, Jeong Dong, E-mail: jdbahk@gnu.ac.kr [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Heo, Jae Bok, E-mail: jbheo72@dau.ac.kr [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of)

2013-05-17

Highlights: •OsRab11 interacts with OsOPR8. •OsOPR8 is localized in the cytosol and peroxisome. •OsRab11 enhances the NADPH consumption by OsOPR8. •Transgenic Arabidopsis overexpressing OsRab11 represents a pathogen-resistant phenotype. -- Abstract: Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Science.gov (United States)

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

2018-01-01

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

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Directory of Open Access Journals (Sweden)

Meraj A. Khan

2018-02-01

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

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

Directory of Open Access Journals (Sweden)

R.B. Oliveira

1998-09-01

Full Text Available The effects of methylmercury (MeHg on histochemical demonstration of the NADPH-diaphorase (NADPH-d activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 µg MeHg and 100 g fish containing 0.03 µg MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 µg/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1 and the other animals were perfused 6 months later (group 2. After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80. Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P<0.05. These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions

NARCIS (Netherlands)

Glogauer, Michael; Marchal, Christophe C.; Zhu, Fei; Worku, Aelaf; Clausen, Björn E.; Foerster, Irmgard; Marks, Peter; Downey, Gregory P.; Dinauer, Mary; Kwiatkowski, David J.

2003-01-01

Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To

NF-κB in The Mechanism of Brain Edema in Acute Liver Failure: Studies in Transgenic Mice

Science.gov (United States)

Jayakumar, A.R.; Bethea, J.R.; Tong, X.Y.; Gomez, J.; Norenberg, M.D.

2014-01-01

, ammonia significantly increased cell swelling (31.7%) in cultured astrocytes from WT mice and displayed cytological abnormalities. Moreover, we observed a lesser increment in inducible nitric oxide synthase and NADPH oxidase activity (both are also known to be activated by NF-κB and to contribute to astrocyte swelling) in astrocyte cultures from Tg mice treated with ammonia, as compared to ammonia-treated WT mice astrocytes. These findings strongly suggest that activation of NF-κB is a critical factor in the development of astrocyte swelling/brain edema in ALF. PMID:21087666

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

Science.gov (United States)

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

2010-07-01

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

Impaired compensation to femoral artery ligation in diet-induced obese mice is primarily mediated via suppression of collateral growth by Nox2 and p47phox.

Science.gov (United States)

DiStasi, Matthew R; Mund, Julie A; Bohlen, H Glenn; Miller, Steven J; Ingram, David A; Dalsing, Michael C; Unthank, Joseph L

2015-10-01

The present study was undertaken to establish the role of NADPH oxidase (Nox) in impaired vascular compensation to arterial occlusion that occurs in the presence of risk factors associated with oxidative stress. Diet-induced obese (DIO) mice characterized by multiple comorbidities including diabetes and hyperlipidemia were used as a preclinical model. Arterial occlusion was induced by distal femoral artery ligation in lean and DIO mice. Proximal collateral arteries were identified as the site of major (∼70%) vascular resistance to calf perfusion by distal arterial pressures, which decreased from ∼80 to ∼30 mmHg with ligation in both lean and DIO mice. Two weeks after ligation, significant vascular compensation occurred in lean but not DIO mice as evidenced by increased perfusion (147 ± 48% vs. 49 ± 29%) and collateral diameter (151 ± 30% vs. 44 ± 17%). Vascular mRNA expression of p22(phox), Nox2, Nox4, and p47(phox) were all increased in DIO mice. Treatment of DIO mice with either apocynin or Nox2ds-tat or with whole body ablation of either Nox2 or p47(phox) ameliorated the impairment in both collateral growth and hindlimb perfusion. Multiparametric flow cytometry analysis demonstrated elevated levels of circulating monocytes in DIO mice without impaired mobilization and demargination after femoral artery ligation. These results establish collateral resistance as the major limitation to calf perfusion in this preclinical model, demonstrate than monocyte mobilization and demarginatin is not suppressed, implicate Nox2-p47(phox) interactions in the impairment of vascular compensation to arterial occlusion in DIO mice, and suggest that selective Nox component suppression/inhibition may be effective as either primary or adjuvant therapy for claudicants. Copyright © 2015 the American Physiological Society.

Therapeutic cloning in individual parkinsonian mice

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Tabar, Viviane; Tomishima, Mark; Panagiotakos, Georgia; Wakayama, Sayaka; Menon, Jayanthi; Chan, Bill; Mizutani, Eiji; Al-Shamy, George; Ohta, Hiroshi; Wakayama, Teruhiko; Studer, Lorenz

2009-01-01

Cell transplantation with embryonic stem (ES) cell progeny requires immunological compatibility with host tissue. ‘Therapeutic cloning’ is a strategy to overcome this limitation by generating nuclear transfer (nt)ES cells that are genetically matched to an individual. Here we establish the feasibility of treating individual mice via therapeutic cloning. Derivation of 187 ntES cell lines from 24 parkinsonian mice, dopaminergic differentiation, and transplantation into individually matched host mice showed therapeutic efficacy and lack of immunological response. PMID:18376409

The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

International Nuclear Information System (INIS)

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

2006-01-01

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

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

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Xiaohui eLi

2015-06-01

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

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

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Piotr Mydel

2006-07-01

Full Text Available The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

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Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

2012-08-01

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

Dynamic flux balancing elucidates NAD(P)H production as limiting response to furfural inhibition in Saccharomyces cerevisiae.

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Pornkamol, Unrean; Franzen, Carl J

2015-08-01

Achieving efficient and economical lignocellulose-based bioprocess requires a robust organism tolerant to furfural, a major inhibitory compound present in lignocellulosic hydrolysate. The aim of this study was to develop a model that could generate quantitative descriptions of cell metabolism for elucidating the cell's adaptive response to furfural. Such a modelling tool could provide strategies for the design of more robust cells. A dynamic flux balance (dFBA) model of Saccharomyces cerevisiae was created by coupling a kinetic fermentation model with a previously published genome-scale stoichiometric model. The dFBA model was used for studying intracellular and extracellular flux responses to furfural perturbations under steady state and dynamic conditions. The predicted effects of furfural on dynamic flux profiles agreed well with previously published experimental results. The model showed that the yeast cell adjusts its metabolism in response to furfural challenge by increasing fluxes through the pentose phosphate pathway, TCA cycle, and proline and serine biosynthesis in order to meet the high demand of NAD(P)H cofactors. The model described here can be used to aid in systematic optimization of the yeast, as well as of the fermentation process, for efficient lignocellulosic ethanol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

OpenAIRE

Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

2008-01-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expressio...

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

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Zeynab Nayernia

2017-10-01

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

The Synergistic Effect of Valsartan and LAF237 [(S-1-[(3-Hydroxy-1-AdamantylAmmo]acetyl-2-Cyanopyrrolidine] on Vascular Oxidative Stress and Inflammation in Type 2 Diabetic Mice

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Min Shen

2012-01-01

Full Text Available Aim. To investigate the combination effects and mechanisms of valsartan (angiotensin II type 1 receptor blocker and LAF237 (DPP-IV inhibitor on prevention against oxidative stress and inflammation injury in db/db mice aorta. Methods. Db/db mice (n=40 were randomized to receive valsartan, LAF237, valsartan plus LAF237, or saline. Oxidative stress and inflammatory reaction in diabetic mice aorta were examined. Results. Valsartan or LAF237 pretreatment significantly increased plasma GLP-1 expression, reduced apoptosis of endothelial cells isolated from diabetic mice aorta. The expression of NAD(PH oxidase subunits also significantly decreased resulting in decreased superoxide production and ICAM-1 (fold change: valsartan : 7.5 ± 0.7, P<0.05; LAF237: 10.2 ± 1.7, P<0.05, VCAM-1 (fold change: valsartan : 5.2 ± 1.2, P<0.05; LAF237: 4.8 ± 0.6, P<0.05, and MCP-1 (fold change: valsartan: 3.2 ± 0.6, LAF237: 4.7 ± 0.8; P<0.05 expression. Moreover, the combination treatment with valsartan and LAF237 resulted in a more significant increase of GLP-1 expression. The decrease of the vascular oxidative stress and inflammation reaction was also higher than monotherapy with valsartan or LAF237. Conclusion. These data indicated that combination treatment with LAF237 and valsartan acts in a synergistic manner on vascular oxidative stress and inflammation in type 2 diabetic mice.

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

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

2017-12-01

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

Impact of angiotensin II on skeletal muscle metabolism and function in mice: contribution of IGF-1, Sirtuin-1 and PGC-1α.

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Kackstein, Katharina; Teren, Andrej; Matsumoto, Yasuharu; Mangner, Norman; Möbius-Winkler, Sven; Linke, Axel; Schuler, Gerhard; Punkt, Karla; Adams, Volker

2013-05-01

Activation of the renin-angiotensin-aldosterone system and increased levels of angiotensin II (Ang-II) occurs in numerous cardiovascular diseases such as chronic heart failure (CHF). Another hallmark in CHF is a reduced exercise tolerance with impaired skeletal muscle function. The aim of this study was to investigate in an animal model the impact of Ang-II on skeletal muscle function and concomitant molecular alterations. Mice were infused with Ang-II for 4 weeks. Subsequently, skeletal muscle function of the soleus muscle was assessed. Expression of selected proteins was quantified by qRT-PCR and Western blot. Infusion of Ang-II resulted in a 33% reduction of contractile force, despite a lack of changes in muscle weight. At the molecular level an increased expression of NAD(P)H oxidase and a reduced expression of Sirt1, PGC-1α and IGF-1 were noticed. No change was evident for the ubiquitin E3-ligases MuRF1 and MafBx and α-sarcomeric actin expression. Cytophotometrical analysis of the soleus muscle revealed a metabolic shift toward a glycolytic profile. This study provides direct evidence of Ang-II-mediated, metabolic deterioration of skeletal muscle function despite preserved muscle mass. One may speculate that the Ang-II-mediated loss of muscle force is due to an activation of NAD(P)H oxidase expression and a subsequent ROS-induced down regulation of IGF-1, PGC-1α and Sirt1. Copyright © 2012 Elsevier GmbH. All rights reserved.

Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs.

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Sundarraj, Kiruthika; Raghunath, Azhwar; Panneerselvam, Lakshmikanthan; Perumal, Ekambaram

2017-02-15

With increased industrial utilization of iron oxide nanoparticles (Fe 2 O 3 -NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe 2 O 3 -NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe 2 O 3 -NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe 2 O 3 -NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe 2 O 3 -NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe 2 O 3 -NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe 2 O 3 -NPs could be an environmental risk factor for reproductive disease. Copyright © 2017 Elsevier Inc. All rights reserved.

The tumor necrosis factor family member TNFSF14 (LIGHT) is required for resolution of intestinal inflammation in mice.

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Krause, Petra; Zahner, Sonja P; Kim, Gisen; Shaikh, Raziyah B; Steinberg, Marcos W; Kronenberg, Mitchell

2014-06-01

The pathogenesis of inflammatory bowel disease (IBD) is associated with a dysregulated mucosal immune response. Expression of the tumor necrosis factor (TNF) superfamily member 14 (TNFSF14, also known as LIGHT [homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes]) on T cells is involved in their activation; transgenic expression of LIGHT on T cells in mice promotes inflammation in multiple organs, including intestine. We investigated the roles for LIGHT in recovery from intestinal inflammation in mice. We studied the role of LIGHT in intestinal inflammation using Tnfsf14(-/-) and wild-type mice. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells into Rag1(-/-) or Tnfsf14(-/-)Rag1(-/-) mice, or by administration of dextran sulfate sodium to Tnfsf14(-/-) or wild-type C57BL/6J mice. Mice were weighed, colon tissues were collected and measured, and histology analyses were performed. We measured infiltrating cell populations and expression of cytokines, chemokines, and LIGHT. After administration of dextran sulfate sodium, Tnfsf14(-/-) mice developed more severe colitis than controls, based on their reduced survival, accelerated loss of body weight, and histologic scores. LIGHT protected mice from colitis via the lymphotoxin β receptor and was expressed mainly by myeloid cells in the colon. Colons of Tnfsf14(-/-) mice also had increased accumulation of innate immune cells and higher levels of cytokines than colons from control mice. LIGHT, therefore, appears to regulate inflammation in the colon. Tnfsf14(-/-) mice develop more severe colitis than control mice. LIGHT signals through the lymphotoxin β receptor in the colon to regulate the innate immune response and mediate recovery from intestinal inflammation. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

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

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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.

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

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Shi, Guang-Xia; Wang, Xue-Rui; Yan, Chao-Qun; He, Tian; Yang, Jing-Wen; Zeng, Xiang-Hong; Xu, Qian; Zhu, Wen; Du, Si-Qi; Liu, Cun-Zhi

2015-12-10

In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

T-cell-dependent control of acute Giardia lamblia infections in mice.

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Singer, S M; Nash, T E

2000-01-01

We have studied immune mechanisms responsible for control of acute Giardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice with G. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to control G. lamblia infections, as has been shown previously for G. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either alphabeta- or gammadelta-T-cell receptor (TCR)-expressing T cells, we show that the alphabeta-TCR-expressing T cells are required to control parasites but that the gammadelta-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection from G. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

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

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

2015-02-01

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

Interaction between the nature of the information and the cognitive requirement of the task in problem solving in mice.

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Wolff, Mathieu; Benhassine, Narimane; Costet, Pierre; Segu, Louis; Buhot, Marie-Christine

2004-11-01

The Morris water maze and the radial-arm maze are two of the most frequently employed behavioral tasks used to assess spatial memory in rodents. In this study, we describe two new behavioral tasks in a radial-arm water maze enabling to combine the advantages of the Morris water maze and the radial-arm maze. In both tasks, spatial and nonspatial learning was assessed and the only task parameter that varied was the nature of the information available which was either spatial (various distal extra-maze cues) or nonspatial (visual intra-maze patterns). In experiment 1, 129T2/Sv mice were able to learn three successive pairwise discriminations [(1) A+/B-, (2) B+/C-, (3) C+/A-] with the same efficiency in both modalities (i.e. spatial and nonspatial modalities). Probe-trials at the end of each of these discriminations revealed particular features of this transverse-patterning-like procedure. In experiment 2, another group of 129T2/Sv mice was submitted to a delayed matching-to-sample working memory task. Mice were able to learn the task and were then able to show resistance to temporal interference as long as 60 min in the spatial modality but they failed to acquire the task in the nonspatial modality. The fact that the nonspatial information was exactly the same in both experiments highlights the existence of an interaction between the cognitive requirements of the task and the nature of the information.

Vagal innervation is required for pulmonary function phenotype in Htr4-/- mice.

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House, John S; Nichols, Cody E; Li, Huiling; Brandenberger, Christina; Virgincar, Rohan S; DeGraff, Laura M; Driehuys, Bastiaan; Zeldin, Darryl C; London, Stephanie J

2017-04-01

Human genome-wide association studies have identified over 50 loci associated with pulmonary function and related phenotypes, yet follow-up studies to determine causal genes or variants are rare. Single nucleotide polymorphisms in serotonin receptor 4 ( HTR4 ) are associated with human pulmonary function in genome-wide association studies and follow-up animal work has demonstrated that Htr4 is causally associated with pulmonary function in mice, although the precise mechanisms were not identified. We sought to elucidate the role of neural innervation and pulmonary architecture in the lung phenotype of Htr4 -/- animals. We report here that the Htr4 -/- phenotype in mouse is dependent on vagal innervation to the lung. Both ex vivo tracheal ring reactivity and in vivo flexiVent pulmonary functional analyses demonstrate that vagotomy abrogates the Htr4 -/- airway hyperresponsiveness phenotype. Hyperpolarized 3 He gas magnetic resonance imaging and stereological assessment of wild-type and Htr4 -/- mice reveal no observable differences in lung volume, inflation characteristics, or pulmonary microarchitecture. Finally, control of breathing experiments reveal substantive differences in baseline breathing characteristics between mice with/without functional HTR4 in breathing frequency, relaxation time, flow rate, minute volume, time of inspiration and expiration and breathing pauses. These results suggest that HTR4's role in pulmonary function likely relates to neural innervation and control of breathing. Copyright © 2017 the American Physiological Society.

Of mice and (Viking?) men: phylogeography of British and Irish house mice.

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Searle, Jeremy B; Jones, Catherine S; Gündüz, Islam; Scascitelli, Moira; Jones, Eleanor P; Herman, Jeremy S; Rambau, R Victor; Noble, Leslie R; Berry, R J; Giménez, Mabel D; Jóhannesdóttir, Fríoa

2009-01-22

The west European subspecies of house mouse (Mus musculus domesticus) has gained much of its current widespread distribution through commensalism with humans. This means that the phylogeography of M. m. domesticus should reflect patterns of human movements. We studied restriction fragment length polymorphism (RFLP) and DNA sequence variations in mouse mitochondrial (mt) DNA throughout the British Isles (328 mice from 105 localities, including previously published data). There is a major mtDNA lineage revealed by both RFLP and sequence analyses, which is restricted to the northern and western peripheries of the British Isles, and also occurs in Norway. This distribution of the 'Orkney' lineage fits well with the sphere of influence of the Norwegian Vikings and was probably generated through inadvertent transport by them. To form viable populations, house mice would have required large human settlements such as the Norwegian Vikings founded. The other parts of the British Isles (essentially most of mainland Britain) are characterized by house mice with different mtDNA sequences, some of which are also found in Germany, and which probably reflect both Iron Age movements of people and mice and earlier development of large human settlements. MtDNA studies on house mice have the potential to reveal novel aspects of human history.

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

Science.gov (United States)

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

2015-09-01

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

Euthanasia of neonatal mice with carbon dioxide

Science.gov (United States)

Pritchett, K.; Corrow, D.; Stockwell, J.; Smith, A.

2005-01-01

Exposure to carbon dioxide (CO2) is the most prevalent method used to euthanize rodents in biomedical research. The purpose of this study was to determine the time of CO2 exposure required to euthanize neonatal mice (0 to 10 days old). Multiple groups of mice were exposed to 100% CO 2 for time periods between 5 and 60 min. Mice were placed in room air for 10 or 20 min after CO2 exposure, to allow for the chance of recovery. If mice recovered at one time point, a longer exposure was examined. Inbred and outbred mice were compared. Results of the study indicated that time to death varied with the age of the animals and could be as long as 50 min on the day of birth and differed between inbred and outbred mice. Institutions euthanizing neonatal mice with CO2 may wish to adjust their CO 2 exposure time periods according the age of the mice and their genetic background. Copyright 2005 by the American Association for Laboratory Animal Science.

Do diosgenin ameliorate urinary bladder toxic effect of ...

African Journals Online (AJOL)

SWEET

2012-01-26

Jan 26, 2012 ... experimental animal models? ... BSO doses using a Swiss albino mouse model. Toxicity modulation ... bladder inflammation induced by CP in rats and mice .... 0.1 ml NADPH (nicotinamide adenine dinucleotide phosphate.

Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

Science.gov (United States)

Wolf, Gerald; Engelmann, Mario; Richter, Karin

2005-01-01

Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…

The dietary polysaccharide maltodextrin promotes Salmonella survival and mucosal colonization in mice.

Directory of Open Access Journals (Sweden)

Kourtney P Nickerson

Full Text Available 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.

Production of knock-in mice in a single generation from embryonic stem cells.

Science.gov (United States)

Ukai, Hideki; Kiyonari, Hiroshi; Ueda, Hiroki R

2017-12-01

The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to ∼3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.

Site requirements and kinetics of immune-dependent elimination of intravascularly administered lung stage schistosomula in mice immunized with highly irradiated cercariae of Schistosoma mansoni

International Nuclear Information System (INIS)

Mangold, B.L.; Dean, D.A.; Coulson, P.S.; Wilson, R.A.

1986-01-01

Experiments were performed to compare the migration and survival of 75Se-labeled schistosomes, introduced by percutaneous cercarial exposure or by intravascular administration of 7-day-old lung stage schistosomula, in control and irradiated cercaria-immunized mice. Schistosomula were intravascularly introduced into the lungs, systemic organs and liver by injection via the femoral vein (FV), left ventricle (LV), and superior mesenteric vein (SMV), respectively. The fate of challenge larvae was examined by autoradiography of host tissues and by recovery of adult worms. It was found that both normal and immune elimination were site-dependent. In control mice 45%-60% of cercarial penetrants and lung schistosomula injected into the FV and LV were recoverable as adult worms, while a significantly greater number (70%-85%) were recoverable when lung schistosomula were injected into the SMV. In immunized mice, parasites introduced as either cercariae or FV-injected schistosomula were both highly sensitive to immune elimination. LV-injected schistosomula were also sensitive but to a slightly lesser degree. In contrast, schistosomula placed directly in the liver by SMV injection were totally insensitive to immune elimination. It was concluded that elimination of schistosomula in irradiated cercaria-immunized mice occurs in the lungs and/or in the systemic organs, but not in the liver. Also, it was concluded that immune elimination is not a rapid process, since more than 7 days were required after intravascular challenge for the development of demonstrable differences between control and immunized mice

Regressive Effect of Myricetin on Hepatic Steatosis in Mice Fed a High-Fat Diet

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Shu-Fang Xia

2016-12-01

Full Text Available Myricetin is an effective antioxidant in the treatment of obesity and obesity-related metabolic disorders. The objective of this study was to explore the regressive effect of myricetin on pre-existing hepatic steatosis induced by high-fat diet (HFD. C57BL/6 mice were fed either a standard diet or a HFD for 12 weeks and then half of the mice were treated with myricetin (0.12% in the diet, w/w while on their respective diets for further 12 weeks. Myricetin treatment significantly alleviated HFD-induced steatosis, decreased hepatic lipid accumulation and thiobarbituric acid reactive substance (TBARS levels, and increased antioxidative enzyme activities, including catalase (CAT, superoxide dismutase (SOD, and glutathione peroxidase (GPx activities. Microarray analysis of hepatic gene expression profiles showed that myricetin significantly altered the expression profiles of 177 genes which were involved in 12 biological pathways, including the peroxisome proliferator activated receptor (PPAR signaling pathway and peroxisome. Further research indicated that myricetin elevated hepatic nuclear Nrf2 translocation, increased the protein expression of heme oxygenase-1 (HO-1 and NAD(PH quinone dehydrogenase 1 (NQO1, reduced the protein expression of PPARγ, and normalized the expressions of genes that were involved in peroxisome and the PPAR signaling pathway. Our data indicated that myricetin might represent an effective therapeutic agent to treat HFD-induced hepatic steatosis via activating the Nrf2 pathway and the PPAR signaling pathway.

A novel Nrf2 activator from microbial transformation inhibits radiation-induced dermatitis in mice

International Nuclear Information System (INIS)

Nakagami, Yasuhiro; Masuda, Kayoko

2016-01-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that regulates many antioxidants, and we have recently succeeded in obtaining a novel Nrf2 activator, RS9, from microbial transformation. RS9 is categorized as a triterpenoid, and well-known triterpenoids such as RTA 402 (bardoxolone methyl) and RTA 408 have been tested in clinical trials. RTA 408 lotion is currently being tested in patients at risk for radiation dermatitis. This prompted us to study the profiles of RS9 in the skin. All the above triterpenoids increased the level of an Nrf2-targeted gene, NADPH:quinone oxidoreductase-1, in normal human epidermal keratinocytes. Among them, the activity of RS9 was prominent; furthermore, the cellular toxicity was less compared with RTA compounds. BALB/c mice were irradiated with 30 Gy/day on Day 0, and compounds were topically applied on the back once daily from Day 1 to Day 30. Dermatitis scores peaked on Day 18, with a score of 2.6 in vehicle-treated mice, and topical applications of 0.1% RTA 402, RTA 408 and RS9 reduced the scores to 1.8, 2.0 and 1.4, respectively. Moreover, the percentage of animals with scores ≥2 was analyzed, and 0.1% RS9 suppressed the percentage from 100% to 47%. These results imply that RS9 has potential efficacy for treating radiation dermatitis.

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

Science.gov (United States)

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

2010-01-01

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

State machine operation of the MICE cooling channel

International Nuclear Information System (INIS)

Hanlet, Pierrick

2014-01-01

The Muon Ionization Cooling Experiment (MICE) is a demonstration experiment to prove the feasibility of cooling a beam of muons for use in a Neutrino Factory and/or Muon Collider. The MICE cooling channel is a section of a modified Study II cooling channel which will provide a 10% reduction in beam emittance. In order to ensure a reliable measurement, MICE will measure the beam emittance before and after the cooling channel at the level of 1%, a relative measurement of 0.001. This renders MICE a precision experiment which requires strict controls and monitoring of all experimental parameters in order to control systematic errors. The MICE Controls and Monitoring system is based on EPICS and integrates with the DAQ, Data monitoring systems, and a configuration database. The cooling channel for MICE has between 12 and 18 superconductnig solenoid coils in 3 to 7 magnets, depending on the staged development of the experiment. The magnets are coaxial and in close proximity which requires coordinated operation of the magnets when ramping, responding to quench conditions, and quench recovery. To reliably manage the operation of the magnets, MICE is implementing state machines for each magnet and an over-arching state machine for the magnets integrated in the cooling channel. The state machine transitions and operating parameters are stored/restored to/from the configuration database and coupled with MICE Run Control. Proper implementation of the state machines will not only ensure safe operation of the magnets, but will help ensure reliable data quality. A description of MICE, details of the state machines, and lessons learned from use of the state machines in recent magnet training tests will be discussed.

Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

Science.gov (United States)

Machida, Takeshi; Ishibashi, Akiko; Kirino, Ai; Sato, Jun-ichi; Kawasaki, Shinji; Niimura, Youichi; Honjoh, Ken-ichi; Miyamoto, Takahisa

2012-01-01

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances. PMID:23029353

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

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Qi-tao Huang

2015-03-01

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

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

Science.gov (United States)

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

2015-01-01

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

Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

Science.gov (United States)

Bedrosian, Andrea S.; Nguyen, Andrew H.; Hackman, Michael; Connolly, Michael K.; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E.; Henning, Justin R.; Barilla, Rocky; Rehman, Adeel; Pachter, H. Leon; Medina-Zea, Marco V.; Cohen, Steven M.; Frey, Alan B.; Acehan, Devrim; Miller, George

2011-01-01

Background & Aims Acute pancreatitis increases morbidity and mortality from organ necrosis by mechanisms that are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. Methods Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier analysis. Results Numbers of MHC II+CD11c+DC increased 100-fold in pancreas of mice with acute pancreatitis, to account for nearly 15% of intra-pancreatic leukocytes. Intra-pancreatic DC acquired an immune phenotype in mice with acute pancreatitis; they expressed higher levels of MHC II and CD86 and increased production of interleukin-6, membrane cofactor protein (MCP)-1, and tumor necrosis factor (TNF)-α. However, rather than inducing an organ-destructive inflammatory process, DC were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DC and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DC died from acinar cell death within 4 days. Depletion of DC from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DC did not require infiltrating neutrophils, activation of NF-κB, or signaling by mitogen-activated protein kinase or TNF-α. Conclusions DC are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress. PMID:21801698

The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice.

Science.gov (United States)

Lamousé-Smith, Esi S; Tzeng, Alice; Starnbach, Michael N

2011-01-01

The presence of a complex and diverse intestinal flora is functionally important for regulating intestinal mucosal immune responses. However, the extent to which a balanced intestinal flora regulates systemic immune responses is still being defined. In order to specifically examine whether the acquisition of a less complex flora influences responses to immunization in the pre-weaning stages of life, we utilize a model in which infant mice acquire an intestinal flora from their mothers that has been altered by broad-spectrum antibiotics. In this model, pregnant dams are treated with a cocktail of antibiotics that alters both the density and microbial diversity of the intestinal flora. After challenge with a subcutaneous immunization, the antibiotic altered flora infant mice have lower antigen specific antibody titers compared to control age-matched mice. In a second model, we examined germ free (GF) mice to analyze how the complete lack of flora influences the ability to mount normal antibody responses following subcutaneous immunization. GF mice do not respond well to immunization and introduction of a normal flora into GF mice restores the capacity of these mice to respond. These results indicate that a gastrointestinal flora reduced in density and complexity at critical time points during development adversely impacts immune responses to systemic antigens.

Expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is increased in the endometrium of women with endometrial cancer and women with polycystic ovary syndrome

DEFF Research Database (Denmark)

Atiomo, William; Shafiee, Mohamad Nasir; Chapman, Caroline

2017-01-01

of differentially expressed genes identified by RNA sequencing, including NAD(P)H quinone dehydrogenase 1 (NQO1), was validated by quantitative reverse transcriptase PCR validation (n = 76) and in the cancer genome atlas UCEC (uterine corpus endometrioid carcinoma) RNA sequencing data set (n = 381). The expression...

Travel Experience in hotels for the MICE Industry

OpenAIRE

Gurkina, Anastasija

2013-01-01

The following report is a research-oriented bachelor thesis that investigates the travel experience in the business oriented concentrated in MICE Industry. The objective of this thesis was to assess and measure the level of satisfaction of the MICE travellers in hotels, as well as to find out what are the services and aspects of the hotel that influences the most on the travel experience. The MICE travellers due to the purposes of their visit require specific services from the hotel t...

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production

DEFF Research Database (Denmark)

Thykær, Jette; Kildegaard, Kanchana Rueksomtawin; Noorman, H.

2008-01-01

was detected in either of the Delta gdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed......The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA...... continued beta-lactam production for the reference strains whereas the Delta gdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results...

Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice

Science.gov (United States)

Smeland, Olav B; Hadera, Mussie G; McDonald, Tanya S; Sonnewald, Ursula; Borges, Karin

2013-01-01

Although certain metabolic characteristics such as interictal glucose hypometabolism are well established for temporal lobe epilepsy (TLE), its pathogenesis still remains unclear. Here, we performed a comprehensive study of brain metabolism in a mouse model of TLE, induced by pilocarpine–status epilepticus (SE). To investigate glucose metabolism, we injected mice 3.5–4 weeks after SE with [1,2-13C]glucose before microwave fixation of the head. Using 1H and 13C nuclear magnetic resonance spectroscopy, gas chromatography—mass spectrometry and high-pressure liquid chromatography, we quantified metabolites and 13C labeling in extracts of cortex and hippocampal formation (HF). Hippocampal levels of glutamate, glutathione and alanine were decreased in pilocarpine–SE mice compared with controls. Moreover, the contents of N-acetyl aspartate, succinate and reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H were decreased in HF indicating impairment of mitochondrial function. In addition, the reduction in 13C enrichment of hippocampal citrate and malate suggests decreased tricarboxylic acid (TCA) cycle turnover in this region. In cortex, we found reduced 13C labeling of glutamate, glutamine and aspartate via the pyruvate carboxylation and pyruvate dehydrogenation pathways, suggesting slower turnover of these amino acids and/or the TCA cycle. In conclusion, mitochondrial metabolic dysfunction and altered amino-acid metabolism is found in both cortex and HF in this epilepsy model. PMID:23611869

Transgenic HFE-dependent induction of hepcidin in mice does not require transferrin receptor-2.

Science.gov (United States)

Schmidt, Paul J; Fleming, Mark D

2012-06-01

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2(Y245X/Y245X) mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, coimmunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner. Copyright © 2012 Wiley Periodicals, Inc.

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

NARCIS (Netherlands)

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

2006-01-01

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

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

Directory of Open Access Journals (Sweden)

Yi-Hsuan Wu

2015-12-01

Full Text Available Glucose-6-phosphate dehydrogenase (G6PD-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α and GTPase myxovirus resistance 1 (MX1—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E and enterovirus 71 (EV71 infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

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

Science.gov (United States)

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

2017-10-01

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

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

Science.gov (United States)

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

2016-10-01

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

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

DEFF Research Database (Denmark)

Bavishi, Krutika; Laursen, Tomas; Martinez, Karen Laurence

2016-01-01

Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially...... was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions...

Pion contamination in the MICE muon beam

International Nuclear Information System (INIS)

Adams, D.; Barclay, P.; Bayliss, V.; Brashaw, T.W.; Alekou, A.; Apollonio, M.; Barber, G.; Asfandiyarov, R.; Blondel, A.; De Bari, A.; Bayes, R.; Bertoni, R.; Bonesini, M.; Blackmore, V.J.; Blot, S.; Bogomilov, M.; Booth, C.N.; Bowring, D.; Boyd, S.; Bravar, U.

2016-01-01

The international Muon Ionization Cooling Experiment (MICE) will perform a systematic investigation of ionization cooling with muon beams of momentum between 140 and 240 MeV/c at the Rutherford Appleton Laboratory ISIS facility. The measurement of ionization cooling in MICE relies on the selection of a pure sample of muons that traverse the experiment. To make this selection, the MICE Muon Beam is designed to deliver a beam of muons with less than ∼1% contamination. To make the final muon selection, MICE employs a particle-identification (PID) system upstream and downstream of the cooling cell. The PID system includes time-of-flight hodoscopes, threshold-Cherenkov counters and calorimetry. The upper limit for the pion contamination measured in this paper is f π  < 1.4% at 90% C.L., including systematic uncertainties. Therefore, the MICE Muon Beam is able to meet the stringent pion-contamination requirements of the study of ionization cooling

Pion contamination in the MICE muon beam

CERN Document Server

Bogomilov, M.; Vankova-Kirilova, G.; Bertoni, R.; Bonesini, M.; Chignoli, F.; Mazza, R.; Palladino, V.; de Bari, A.; Cecchet, G.; Capponi, M.; Iaciofano, A.; Orestano, D.; Pastore, F.; Tortora, L.; Kuno, Y.; Sakamoto, H.; Ishimoto, S.; Japan, Ibaraki; Filthaut, F.; Hansen, O.M.; Ramberger, S.; Vretenar, M.; Asfandiyarov, R.; Blondel, A.; Drielsma, F.; Karadzhov, Y.; Charnley, G.; Collomb, N.; Gallagher, A.; Grant, A.; Griffiths, S.; Hartnett, T.; Martlew, B.; Moss, A.; Muir, A.; Mullacrane, I.; Oates, A.; Owens, P.; Stokes, G.; Warburton, P.; White, C.; Adams, D.; Barclay, P.; Bayliss, V.; Bradshaw, T.W.; Courthold, M.; Francis, V.; Fry, L.; Hayler, T.; Hills, M.; Lintern, A.; Macwaters, C.; Nichols, A.; Preece, R.; Ricciardi, S.; Rogers, C.; Stanley, T.; Tarrant, J.; Watson, S.; Wilson, A.; Bayes, R.; Nugent, J.C.; Soler, F.J.P.; Cooke, P.; Gamet, R.; Alekou, A.; Apollonio, M.; Barber, G.; Colling, D.; Dobbs, A.; Dornan, P.; Hunt, C.; Lagrange, J-B.; Long, K.; Martyniak, J.; Middleton, S.; Pasternak, J.; Santos, E.; Savidge, T.; Uchida, M.A.; Blackmore, V.J.; Carlisle, T.; Cobb, J.H.; Lau, W.; Rayner, M.A.; Tunnell, C.D.; Booth, C.N.; Hodgson, P.; Langlands, J.; Nicholson, R.; Overton, E.; Robinson, M.; Smith, P.J.; Dick, A.; Ronald, K.; Speirs, D.; Whyte, C.G.; Young, A.; Boyd, S.; Franchini, P.; Greis, J.R.; Pidcott, C.; Taylor, I.; Gardener, R.; Kyberd, P.; Littlefield, M.; Nebrensky, J.J.; Bross, A.D.; Fitzpatrick, T.; Leonova, M.; Moretti, A.; Neuffer, D.; Popovic, M.; Rubinov, P.; Rucinski, R.; Roberts, T.J.; Bowring, D.; DeMello, A.; Gourlay, S.; Li, D.; Prestemon, S.; Virostek, S.; Zisman, M.; Drews, M.; Hanlet, P.; Kafka, G.; Kaplan, D.M.; Rajaram, D.; Snopok, P.; Torun, Y.; Winter, M.; Blot, S.; Kim, Y.K.; Bravar, U.; Onel, Y.; Cremaldi, L.M.; Hart, T.L.; Luo, T.; Sanders, D.A.; Summers, D.J.; Cline, D.; Yang, X.; Coney, L.; Hanson, G.G.; Heidt, C.

2016-01-01

The international Muon Ionization Cooling Experiment (MICE) will perform a systematic investigation of ionization cooling with muon beams of momentum between 140 and 240\\,MeV/c at the Rutherford Appleton Laboratory ISIS facility. The measurement of ionization cooling in MICE relies on the selection of a pure sample of muons that traverse the experiment. To make this selection, the MICE Muon Beam is designed to deliver a beam of muons with less than $\\sim$1\\% contamination. To make the final muon selection, MICE employs a particle-identification (PID) system upstream and downstream of the cooling cell. The PID system includes time-of-flight hodoscopes, threshold-Cherenkov counters and calorimetry. The upper limit for the pion contamination measured in this paper is $f_\\pi < 1.4\\%$ at 90\\% C.L., including systematic uncertainties. Therefore, the MICE Muon Beam is able to meet the stringent pion-contamination requirements of the study of ionization cooling.

Change of the NADPH depending superoxide producing and ferri hemoglobin reducing activities of cytochrome b558 from spleen cells and erythrocytes membranes induced by the radiation of different character

International Nuclear Information System (INIS)

Melkonyan, L.G.; Simonyan, R.M.; Simonyan, M.A.; Sekoyan, E.S.

2009-01-01

After the X radiation, UVA radiation and ultrasound radiation of new isoforms of cytochrome cyt b 5 58 from rats erythrocyte membranes - EM (cyt b 5 58III) and from spleen cell membranes (SCM) in vitro, as well as after the radiation of EM ex vivo, the suppression of both NADPH depending O 2 - producing and ferrihemoglobin (ferriHb)-reducing activities of cyt b 5 58 from EM and SCM in homogeneous (in solution) and heterogeneous phases (in EM and SCM) at various scopes takes place. These changes are associated with the destabilization of EM and SCM, conditioned by the change of the aggregation degree of these hemoproteins in EM and SCM, hemoproteins as a result of the influence of the hydrogen peroxide formed during radiolysis and photolysis of the water medium. After He-Ne laser radiation of the cyt b 5 58 from EM and SCM in vitro an increase of the NADPH depending O 2 - producing and ferriHb-reducing activities of the cyt b 5 58 from EM and SCM in homogenous and heterogeneous phases (in membranes) takes place. It is supposed that the suppression (by X-, UVA- and US-radiation) and the stimulation (by He-Ne laser radiation) of the immune system activity and the oxygen homeostasis are associated with the corresponding decrease and increase of the NADPH depending O 2 - producing and ferriHb-reducings activity of the new isoforms of cyt b 5 58 from EM and SCM in homogeneous and heterogeneous phases

Decoding NADPH oxidase 4 expression in human tumors

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Jennifer L. Meitzler

2017-10-01

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

Piper betle Induced Cytoprotective Genes and Proteins via the Nrf2/ARE Pathway in Aging Mice.

Science.gov (United States)

Aliahmat, Nor Syahida; Abdul Sani, Nur Fathiah; Wan Hasan, Wan Nuraini; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2016-01-01

The objective of this study was to elucidate the underlying antioxidant mechanism of aqueous extract of Piper betle (PB) in aging rats. The nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE pathway involving phase II detoxifying and antioxidant enzymes plays an important role in the antioxidant system by reducing electrophiles and reactive oxygen species through induction of phase II enzymes and proteins. Genes and proteins of phase II detoxifying antioxidant enzymes were analyzed by QuantiGenePlex 2.0 Assay and Western blot analysis. PB significantly induced genes and proteins of phase II and antioxidant enzymes, NAD(P)H quinone oxidoreductase 1, and catalase in aging mice (p < 0.05). The expression of these enzymes were stimulated via translocation of Nrf2 into the nucleus, indicating the involvement of ARE, a cis-acting motif located in the promoter region of nearly all phase II genes. PB was testified for the first time to induce cytoprotective genes through the Nrf2/ARE signaling pathway, thus unraveling the antioxidant mechanism of PB during the aging process. © 2016 S. Karger AG, Basel.

Impaired Muscle Regeneration in Ob/ob and Db/db Mice

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Mai-Huong Nguyen

2011-01-01

Full Text Available In obesity and type 2 diabetes, efficient skeletal muscle repair following injury may be required, not only for restoring muscle structure and function, but also for maintaining exercise capacity and insulin sensitivity. The hypothesis of this study was that muscle regeneration would be impaired in ob/ob and db/db mice, which are common mouse models of obesity and type 2 diabetes. Muscle injury was produced by cardiotoxin injection, and regeneration was assessed by morphological and immunostaining techniques. Muscle regeneration was delayed in ob/ob and db/db mice, but not in a less severe model of insulin resistance – feeding a high-fat diet to wild-type mice. Angiogenesis, cell proliferation, and myoblast accumulation were also impaired in ob/ob and db/db mice, but not the high-fat diet mice. The impairments in muscle regeneration were associated with impaired macrophage accumulation; macrophages have been shown previously to be required for efficient muscle regeneration. Impaired regeneration in ob/ob and db/db mice could be due partly to the lack of leptin signaling, since leptin is expressed both in damaged muscle and in cultured muscle cells. In summary, impaired muscle regeneration in ob/ob and db/db mice was associated with reduced macrophage accumulation, angiogenesis, and myoblast activity, and could have implications for insulin sensitivity in the skeletal muscle of obese and type 2 diabetic patients.

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

Science.gov (United States)

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

2017-11-01

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

Compensatory eye movements in mice

NARCIS (Netherlands)

A.M. van Alphen (Arjan)

2002-01-01

textabstractThis thesis will address the generation of compensatory eye movements in naturally mutated or genetically modified mice. The reason for generating compensatory eye movements is solely related to the requirements for good vision. In a subject moving through its environment the projection

Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice.

Directory of Open Access Journals (Sweden)

Amanda J Beck

Full Text Available Duchenne muscular dystrophy (DMD is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin. In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.

Prenatal administration of the cytochrome P4501A inducer, Β-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: Implications for bronchopulmonary dysplasia (BPD) in premature infants

International Nuclear Information System (INIS)

Couroucli, Xanthi I.; Liang Yanhong Wei; Jiang Weiwu; Wang Lihua; Barrios, Roberto; Yang Peiying; Moorthy, Bhagavatula

2011-01-01

Supplemental oxygen contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this investigation, we tested the hypothesis that prenatal treatment of pregnant mice (C57BL/6J) with the cytochrome P450 (CYP)1A1 inducer, ss-napthoflavone (BNF), will lead to attenuation of lung injury in newborns (delivered from these dams) exposed to hyperoxia by mechanisms entailing transplacental induction of hepatic and pulmonary CYP1A enzymes. Pregnant mice were administered the vehicle corn oil (CO) or BNF (40 mg/kg), i.p., once daily for 3 days on gestational days (17-19), and newborns delivered from the mothers were either maintained in room air or exposed to hyperoxia (> 95% O 2 ) for 1-5 days. After 3-5 days of hyperoxia, the lungs of CO-treated mice showed neutrophil infiltration, pulmonary edema, and perivascular inflammation. On the other hand, BNF-pretreated neonatal mice showed decreased susceptibility to hyperoxic lung injury. These mice displayed marked induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities, and levels of the corresponding apoproteins and mRNA levels until PND 3 in liver, while CYP1A1 expression alone was augmented in the lung. Prenatal BNF did not significantly alter gene expression of pulmonary NAD(P)H quinone reductase (NQO1). Hyperoxia for 24-72 h resulted in increased pulmonary levels of the F 2 -isoprostane 8-iso-PGF 2α , whose levels were decreased in mice prenatally exposed to BNF. In conclusion, our results suggest that prenatal BNF protects newborns against hyperoxic lung injury, presumably by detoxification of lipid hydroperoxides by CYP1A enzymes, a phenomenon that has implications for prevention of BPD in infants. - Highlights: → Supplemental oxygen is routinely administered to premature infants. → Hyperoxia causes lung injury in experimental animals. → Prenatal treatment of mice with beta-naphthoflavone attenuates oxygen injury �

Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum

Science.gov (United States)

Hinova-Palova, Dimka V.; Edelstein, Lawrence; Landzhov, Boycho; Minkov, Minko; Malinova, Lina; Hristov, Stanislav; Denaro, Frank J.; Alexandrov, Alexandar; Kiriakova, Teodora; Brainova, Ilina; Paloff, Adrian; Ovtscharoff, Wladimir

2014-01-01

We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 μm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 μm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 μm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum—one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies. PMID:24904317

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

Directory of Open Access Journals (Sweden)

Dorota Bryk

2017-01-01

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

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

Science.gov (United States)

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

2016-04-15

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

Metabolic pathways leading to detoxification of triptolide, a major active component of the herbal medicine Tripterygium wilfordii.

Science.gov (United States)

Du, Fuying; Liu, Zhaohua; Li, Xinxiu; Xing, Jie

2014-08-01

Triptolide (TP) shows promising anti-inflammatory and antitumor activity but with severe toxicity. TP is a natural reactive electrophile containing three epoxide groups, which are usually linked to hepatotoxicity via their ability to covalently bind to cellular macromolecules. In this study, metabolic pathways leading to detoxification of TP were evaluated in glutathione (GSH)-depleted (treated with L-buthionine-S,R-sulfoxinine, BSO) and aminobenzotriazole (ABT; a non-specific inhibitor for P450s)-treated mice. The toxicity of TP in mice was evaluated in terms of mortality and levels of serum alanine transaminase (ALT). In incubates with NADPH- and GSH-supplemented liver microsomes, seven GSH conjugates derived from TP were detected. In mice, these hydrolytically unstable GSH conjugates underwent γ-glutamyltranspeptidase/dipeptidases-mediated hydrolysis leading to two major cysteinylglycine conjugates, which underwent further hydrolysis by dipeptidases to form two cysteine conjugates of TP. In ABT-treated mice, the hydroxylated metabolites of TP were found at a lower level than normal mice, and their subsequent conjugated metabolites were not found. The level of cysteinylglycine and cysteine conjugates derived from NADPH-independent metabolism increased in mice treated with both TP and BSO (or ABT), which could be the stress response to toxicity of TP. Compared with normal mice, mortality and ALT levels were significantly higher in TP-treated mice, indicating the toxicity of TP. Pretreatment of ABT increased the toxicity caused by TP, whereas the mortality decreased in GSH-depleted mice. Metabolism by cytochrome P450 enzymes to less reactive metabolites implied a high potential for detoxification of TP. The GSH conjugation pathway also contributed to TP's detoxification in mice. Copyright © 2013 John Wiley & Sons, Ltd.

Prodigiosin inhibits gp91phox and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia–ischemia

International Nuclear Information System (INIS)

Chang, Chia-Che; Wang, Yea-Hwey; Chern, Chang-Ming; Liou, Kuo-Tong; Hou, Yu-Chang; Peng, Yu-Ta; Shen, Yuh-Chiang

2011-01-01

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen–glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100 μg/kg, i.v.) at 1 h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91 phox ), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood–brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91 phox and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91 phox and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice. -- Highlights: ► Prodigiosin ameliorated brain infarction and deficits. ► Prodigiosin protected against hypoxia/reperfusion-induced brain injury. ► Prodigiosin diminished oxidative/nitrosativestress and leukocytes infiltration. ► Prodigiosin reduced BBB breakdown. ► Prodigiosin down-regulated gp91 phox and iNOS by inhibiting NF-κB activation.

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

Directory of Open Access Journals (Sweden)

Shin-Ei Cheng

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

Altered pancreatic growth and insulin secretion in WSB/EiJ mice.

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Maggie M Ho

Full Text Available These data suggest that insulin secretion in WSB mice is blunted specifically in vivo, either due to a reduced insulin requirement and/or due to factors that are absent or destroyed in vitro. These studies also highlight the role of post-natal growth in determining adult β-cell mass. Mice are important animal models for the study of metabolic physiology and the genetics of complex traits. Wild-derived inbred mouse strains, such as WSB/EiJ (WSB, are unrelated to the commonly studied mouse strains and are valuable tools to identify novel genes that modify disease risk. We have previously shown that in contrast to C57BL/6J (B6 mice, WSB mice fed a high fat diet do not develop hyperinsulinemia or insulin resistance, and had nearly undetectable insulin secretion in response to an intraperitoneal glucose challenge. As hyperinsulinemia may drive obesity and insulin resistance, we examined whether defects in β-cell mass or function could contribute to the low insulin levels in WSB mice. In young WSB mice, β-cell mass was similar to B6 mice. However, we found that adult WSB mice had reduced β-cell mass due to reduced pancreatic weights. Pancreatic sizes were similar between the strains when normalized to body weight, suggesting their pancreatic size is appropriate to their body size in adults, but overall post-natal pancreatic growth was reduced in WSB mice compared to B6 mice. Islet architecture was normal in WSB mice. WSB mice had markedly increased insulin secretion from isolated islets in vitro. These data suggest that insulin secretion in WSB mice is blunted specifically in vivo, either due to a reduced insulin requirement and/or due to factors that are absent or destroyed in vitro. These studies suggest that WSB mice may provide novel insight into mechanisms regulating insulin secretion and also highlight the role of post-natal growth in determining adult β-cell mass.

A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

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Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

2014-02-28

NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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Wilson Belinda

2008-05-01

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

Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

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Biljana Musicki

Full Text Available Testosterone deficiency is associated with sickle cell disease (SCD, but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH levels compared with wild type (WT mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR, but not cholesterol side-chain cleavage enzyme (P450scc, in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

Periostin is an extracellular matrix protein required for eruption of incisors in mice

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Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira

2006-01-01

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin -/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin -/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone

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

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Veerachat Muangsombut

2017-08-01

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

Linkage disequilibrium in wild mice.

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Cathy C Laurie

2007-08-01

Full Text Available Crosses between laboratory strains of mice provide a powerful way of detecting quantitative trait loci for complex traits related to human disease. Hundreds of these loci have been detected, but only a small number of the underlying causative genes have been identified. The main difficulty is the extensive linkage disequilibrium (LD in intercross progeny and the slow process of fine-scale mapping by traditional methods. Recently, new approaches have been introduced, such as association studies with inbred lines and multigenerational crosses. These approaches are very useful for interval reduction, but generally do not provide single-gene resolution because of strong LD extending over one to several megabases. Here, we investigate the genetic structure of a natural population of mice in Arizona to determine its suitability for fine-scale LD mapping and association studies. There are three main findings: (1 Arizona mice have a high level of genetic variation, which includes a large fraction of the sequence variation present in classical strains of laboratory mice; (2 they show clear evidence of local inbreeding but appear to lack stable population structure across the study area; and (3 LD decays with distance at a rate similar to human populations, which is considerably more rapid than in laboratory populations of mice. Strong associations in Arizona mice are limited primarily to markers less than 100 kb apart, which provides the possibility of fine-scale association mapping at the level of one or a few genes. Although other considerations, such as sample size requirements and marker discovery, are serious issues in the implementation of association studies, the genetic variation and LD results indicate that wild mice could provide a useful tool for identifying genes that cause variation in complex traits.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

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Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

Molecular and functional characterization of ferredoxin NADP(H oxidoreductase from Gracilaria chilensis and its complex with ferredoxin

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María Alejandra Vorphal

Full Text Available Abstract Backgroud Ferredoxin NADP(H oxidoreductases (EC 1.18.1.2 (FNR are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP+ to NADPH using the electrons provided by Ferredoxin (Fd, a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. Methods The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5′RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. Results The kinetic analysis shows KMNADPH of 12.5 M and a kcat of 86 s−1, data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS, sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. Conclusion The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

Sevoflurane Inhalation Accelerates the Long-Term Memory Consolidation via Small GTPase Overexpression in the Hippocampus of Mice in Adolescence.

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Nakamura, Emi; Kinoshita, Hiroyuki; Feng, Guo-Gang; Hayashi, Hisaki; Satomoto, Maiko; Sato, Motohiko; Fujiwara, Yoshihiro

2016-01-01

Sevoflurane exposure impairs the long-term memory in neonates. Whether the exposure to animals in adolescence affects the memory, however, has been unclear. A small hydrolase enzyme of guanosine triphosphate (GTPase) rac1 plays a role in the F-actin dynamics related to the synaptic plasticity, as well as superoxide production via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The current study was designed to examine whether sevoflurane exposure to mice in early adolescence modifies the long-term learning ability concomitantly with the changes in F-actin constitution as well as superoxide production in the hippocampus according to the levels of rac1 protein expression. Four-week-old mice were subjected to the evaluation of long-term learning ability for three days. On day one, each mouse was allowed to enter a dark chamber for five min to acclimatization. On day two, the procedure was repeated with the addition of an electric shock as soon as a mouse entered the dark chamber. All mice subsequently inhaled 2 L/min air with (Sevoflurane group) and without (Control group) 2.5% sevoflurane for three hours. On day three, each mouse was placed on the platform and retention time, which is the latency to enter the dark chamber, was examined. The brain removed after the behavior test, was used for analyses of immunofluorescence, Western immunoblotting and intracellular levels of superoxide. Sevoflurane exposure significantly prolonged retention time, indicating the enhanced long-term memory. Sevoflurane inhalation augmented F-actin constitution coexisting with the rac1 protein overexpression in the hippocampus whereas it did not alter the levels of superoxide. Sevoflurane exposure to 4-week-old mice accelerates the long-term memory concomitantly with the enhanced F-actin constitution coexisting with the small GTPase rac1 overexpression in the hippocampus. These results suggest that sevoflurane inhalation may amplify long-term memory

Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

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Weston, K.M.; Ju, S.T.; Lu, C.Y.; Sy, M.S.

1988-01-01

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication.

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Paul D Bozyk

Full Text Available In bronchopulmonary dysplasia (BPD, alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.

Molecular Determinants of Influenza Virus Pathogenesis in Mice

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Katz, Jaqueline M.; York, Ian A.

2015-01-01

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account. PMID:25038937

Facile photoreduction of graphene oxide by an NAD(P)H model: Hantzsch 1,4-dihydropyridine.

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Zhang, Hui-Hui; Liu, Qiang; Feng, Ke; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

2012-05-29

To make "clean" reduced GO sheets in high quality and in large scale, a natural reduced nicotinamine adenine dinucleotide NAD(P)H model, Hantzsch 1,4-dihydropyridine (HEH), is used as a mild organic photoreductant in this work. Benefiting from the intense absorption of HEH in the range of 300-420 nm, the graphene oxide (GO) can be readily reduced by HEH under UV light irradiation (λ > 320 nm) to afford single or few-layer reduced graphene oxide at room temperature. Studies on reduction extent reveal that both irradiation time and concentration ratio of HEH to GO are important for effective reduction of GO under UV light. The as-prepared photochemically reduced graphene oxide (PRGO) dispersion is stable without the need for any polymeric or surfactant stabilizers. Simply by extraction treatment, the "clean" PRGO sheets can be obtained in large quantities, and its conductivity approaches to 4680 S·m(-1) that is the highest value reported by photochemical approaches so far.

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

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

2008-01-01

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

Reduction of oxidative damages induced by titanium dioxide nanoparticles correlates with induction of the Nrf2 pathway by GSPE supplementation in mice.

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Niu, Linpeng; Shao, Mengjiao; Liu, Yuan; Hu, Jinfeng; Li, Ruofan; Xie, Heran; Zhou, Lixiao; Shi, Lei; Zhang, Rong; Niu, Yujie

2017-09-25

Titanium dioxide nanoparticles (TiO 2 NPs) are widely used to additives in cosmetics, pharmaceuticals, paints and foods. Recent studies have demonstrated that TiO 2 NPs increased the risk of cancer and the mechanism might relate with oxidative stress. Grape seed procyanidin extract (GSPE) is a natural compound which has been demonstrated to possess a wide array of pharmacological and biochemical actions, including anti-inflammatory, anti-carcinogenic, and antioxidant properties. Our data show that GSPE prevents the changes of histopathology and biomarkers in heart, liver and kidney that occur in mice exposed to TiO 2 NPs. After pretreatment with GSPE, the DNA damage, reactive oxygen species (ROS) generation and malondialdehyde (MDA) content in mice exposed to TiO 2 NPs had statistically significant decreases in dose dependent manners. GSPE increased the expression of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), NAD(P)H dehydrogenase[quinine] 1(NQO1), heme oxygenase 1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC). We conclude that grape seed procyanidin extract prevents the majority of tissue and molecular damage resulting from nanoparticle treatment. The protective effect of GSPE may be due to its strong antioxidative activities which related with the activated Nrf2 and its down-regulated genes including NQO1, HO-1 and GCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

NADPH-thioredoxin reductase C mediates the response to oxidative stress and thermotolerance in the cyanobacterium Anabaena sp. PCC7120.

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ANA MARÍA SÁNCHEZ-RIEGO

2016-08-01

Full Text Available NTRC (NADPH-thioredoxin reductase C is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (∆ntrC, apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

The endothelial cell receptor GRP78 is required for mucormycosis pathogenesis in diabetic mice

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Liu, Mingfu; Spellberg, Brad; Phan, Quynh T.; Fu, Yue; Fu, Yong; Lee, Amy S.; Edwards, John E.; Filler, Scott G.; Ibrahim, Ashraf S.

2010-01-01

Mucormycosis is a fungal infection of the sinuses, brain, or lungs that causes a mortality rate of at least 50% despite first-line therapy. Because angioinvasion is a hallmark of mucormycosis infections, we sought to define the endothelial cell receptor(s) for fungi of the order Mucorales (the fungi that cause mucormycosis). Furthermore, since patients with elevated available serum iron, including those with diabetic ketoacidosis (DKA), are uniquely susceptible to mucormycosis, we sought to define the role of iron and glucose in regulating the expression of such a receptor. Here, we have identified glucose-regulated protein 78 (GRP78) as what we believe to be a novel host receptor that mediates invasion and damage of human endothelial cells by Rhizopus oryzae, the most common etiologic species of Mucorales, but not Candida albicans or Aspergillus fumigatus. Elevated concentrations of glucose and iron, consistent with those seen during DKA, enhanced GRP78 expression and the resulting R. oryzae invasion and damage of endothelial cells in a receptor-dependent manner. Mice with DKA, which have enhanced susceptibility to mucormycosis, exhibited increased expression of GRP78 in sinus, lungs, and brain compared with normal mice. Finally, GRP78-specific immune serum protected mice with DKA from mucormycosis. These results suggest a unique susceptibility of patients with DKA to mucormycosis and provide a foundation for the development of new therapeutic interventions for these deadly infections. PMID:20484814

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

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Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P. (MCW); (Charles U); (UTSMC)

2012-03-15

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

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Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

2009-03-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice.

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Leidinger, Charlotte; Herrmann, Felix; Thöne-Reineke, Christa; Baumgart, Nadine; Baumgart, Jan

2017-03-06

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

High-resolution structure of AKR1a4 in the apo form and its interaction with ligands

International Nuclear Information System (INIS)

Faucher, Frédérick; Jia, Zongchao

2012-01-01

Despite its high affinity for NADPH, AKR1a4 crystallized in the apo form, which is very rare for aldo-keto reductase enzymes. Aldo-keto reductase 1a4 (AKR1a4; EC 1.1.1.2) is the mouse orthologue of human aldehyde reductase (AKR1a1), the founding member of the AKR family. As an NADPH-dependent enzyme, AKR1a4 catalyses the conversion of d-glucuronate to l-gulonate. AKR1a4 is involved in ascorbate biosynthesis in mice, but has also recently been found to interact with SMAR1, providing a novel mechanism of ROS regulation by ATM. Here, the crystal structure of AKR1a4 in its apo form at 1.64 Å resolution as well as the characterization of the binding of AKR1a4 to NADPH and P44, a peptide derived from SMAR1, is presented

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

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Williams, Clintoria R; Gooch, Jennifer L

2014-02-21

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

Immune mechanisms in Ehrlich ascites tumor growth in mice

International Nuclear Information System (INIS)

Marusic, M.

1979-01-01

Normal mice immunised with irradiated Ehrlich ascites tumor (EAT) cells rejected EAT challenge given 2 weeks later but T-cell-deficient thymectomised lethally irradiated, and bone-marrow-reconstituted (TIR) mice succumbed. However, when TIR mice were injected i.v. with thymus, lymph node, or spleen cells from normalsyngetic donors immediately following i.p. injection of irradiated EAT cells, they rejected the subsequent tumor challenge. This induction of immunity in TIR mice was shown to be T-cell dependent. Spleen cells from EAT- bearing mice given immediately after irradiated tumor cells were also able to promote rejection of EAT challenge in TIR mice. Spleen cells from EAT-immune mice inhibited EAT growth when admixed with tumor cells prior to i.p. injection into normal recipients, but had no effect on progressive tumor growth when given i.v. immediately after i.p. tumor injection. Immune serum inhibited i.p. EAT growth when given either i.p. or i.v. Whereas inhibition of EAT growth by admixed spleen cells was shown to be T-cell independent. The data indicate that T lymphocytes are required only in the induction phase of the immune reponse of mice against EAT, while the efferent phase of the response is accomplished by serum antibodies, perhaps through an interaction with host macrophages. (author)

Bone growth and turnover in progesterone receptor knockout mice.

Energy Technology Data Exchange (ETDEWEB)

Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O' Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

2008-05-01

The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans.

Science.gov (United States)

Manfrini, Nicola; Ricciardi, Sara; Miluzio, Annarita; Fedeli, Maya; Scagliola, Alessandra; Gallo, Simone; Brina, Daniela; Adler, Thure; Busch, Dirk H; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě de Angelis, Martin; Biffo, Stefano

2017-12-01

Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4 + Effector Memory T cells. In human CD4 + T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4 + T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs

Energy Technology Data Exchange (ETDEWEB)

Sundarraj, Kiruthika; Raghunath, Azhwar; Panneerselvam, Lakshmikanthan; Perumal, Ekambaram, E-mail: ekas2009@buc.edu.in

2017-02-15

With increased industrial utilization of iron oxide nanoparticles (Fe{sub 2}O{sub 3}-NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe{sub 2}O{sub 3}-NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe{sub 2}O{sub 3}-NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe{sub 2}O{sub 3}-NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe{sub 2}O{sub 3}-NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe{sub 2}O{sub 3}-NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe{sub 2}O{sub 3}-NPs could be an environmental risk factor for reproductive disease. - Highlights: • Fe{sub 2}O{sub 3}-NPs caused adverse effects on the seminal vesicle and prostate gland of mice • Heat shock proteins (Hsp60, 70 and 90) were

Identification of the NADPH Oxidase 4 Inhibiting Principle of Lycopus europaeus

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Silvia Revoltella

2018-03-01

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

Factors influencing insulin and glucagon secretion in lean and genetically obese mice

International Nuclear Information System (INIS)

Beloff-Chain, A.; Newman, M.E.; Mansford, K.R.L.

1977-01-01

The control of 125 I-labelled insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antiinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice. (orig./AJ) [de

Factors influencing insulin and glucagon secretion in lean and genetically obese mice

Energy Technology Data Exchange (ETDEWEB)

Beloff-Chain, A; Newman, M E; Mansford, K R.L. [Imperial Coll. of Science and Technology, London (UK). Dept. of Biochemistry

1977-01-01

The control of /sup 125/I-labelled insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antiinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.

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

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Marta Berrocal-Lobo

2010-12-01

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

ADAM10 regulates Notch function in intestinal stem cells of mice.

Science.gov (United States)

Tsai, Yu-Hwai; VanDussen, Kelli L; Sawey, Eric T; Wade, Alex W; Kasper, Chelsea; Rakshit, Sabita; Bhatt, Riha G; Stoeck, Alex; Maillard, Ivan; Crawford, Howard C; Samuelson, Linda C; Dempsey, Peter J

2014-10-01

A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

Science.gov (United States)

Stadler, Krisztian; Bonini, Marcelo G.; Dallas, Shannon; Duma, Danielle; Mason, Ronald P.; Kadiiska, Maria B.

2008-01-01

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression. PMID:18559982

Tissue inhibitor of matrix metalloproteinase-1 is required for high-fat diet-induced glucose intolerance and hepatic steatosis in mice

DEFF Research Database (Denmark)

Fjære, Even; Andersen, Charlotte; Myrmel, Lene Secher

2015-01-01

-induced glucose intolerance and hepatic steatosis using the Timp1 null mice. METHODS: Timp1 knockout (TKO) and wild type (TWT) mice were fed chow, high-fat diet (HFD) or intermediate fat and sucrose diet (IFSD). We determined body weight, body composition, lipid content of the liver, energy intake, energy...... and had lower energy efficiency than TWT mice when fed HFD, but not when fed chow or IFSD. Importantly, TKO mice were protected from development of HFD- as well as IFSD-induced glucose intolerance, hepatic steatosis, and altered expression of genes involved in hepatic lipid metabolism and inflammation....... CONCLUSION: Collectively, our results indicate that TIMP-1 contributes to the development of diet-induced hepatic steatosis and glucose intolerance and may be a potential therapeutic target....

Dose rate effectiveness in radiation-induced teratogenesis in mice

International Nuclear Information System (INIS)

Kato, F.; Ootsuyama, A.; Norimura, T.

2000-01-01

To investigate the role of p53 gene in tissue repair of teratogenic injury, we compared incidence of radiation-induced malformations in homozygous p53(-/-) mice, heterozygous p53(+/-) mice and wild-type p53(+/+) mice. After X-irradiation with 2 Gy at high dose rate on 9.5 days of gestation, p53(-/-) mice showed higher incidences of anomalies and higher resistance to prenatal deaths than p53(+/+) mice. This reciprocal relationship of radiosensitivity to anomalies and deaths supports the notion that embryos or fetuses have a p53-dependent 'guardian' that aborts cells bearing radiation-induced teratogenic DNA damage. In fact, after X-irradiation, the number of apoptotic cells was greatly increased in p53(+/+) fetuses but not in p53(-/-) fetuses. The same dose of γ-ray exposure at low dose rate on 9.5-10.5 day of gestation produced significant reduction of radiation-induced malformation in p53(+/+) and p53(+/-) mice, remained teratogenic for p53(-/-) mice. These results suggest that complete elimination of teratogenic damage from irradiated tissues requires the concerted cooperation of two mechanisms; proficient DNA repair and the p53-dependent apoptotic tissue repair. When concerted DNA repair and apoptosis functions efficiently, there is a threshold dose-rate for radiation-induced malformations. (author)

Ethanol self-administration in serotonin transporter knockout mice: unconstrained demand and elasticity.

Science.gov (United States)

Lamb, R J; Daws, L C

2013-10-01

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self-administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self-administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self-administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Deficiency of Nox2 prevents angiotensin II-induced inward remodeling in cerebral arterioles

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Siu-Lung eChan

2013-06-01

Full Text Available Angiotensin II is an important determinant of inward remodeling in cerebral arterioles. Many of the vascular effects of angiotensin II are mediated by reactive oxygen species generated from homologues of NADPH oxidase with Nox2 predominating in small arteries and arterioles. Therefore, we tested the hypothesis that superoxide generated by Nox2 plays a role in angiotensin II-induced cerebral arteriolar remodeling. We examined Nox2-deficient and wild-type mice in which a pressor or a non-pressor dose of angiotensin II (1000 or 200 ng/kg/day or saline was infused for 4 weeks via osmotic minipumps. Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of cerebral arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area (by histology and superoxide level (by hydroethidine staining of cerebral arterioles were determined ex vivo. The pressor, but not the non-pressor, dose of angiotensin II significantly increased systolic arterial pressure in both wild-type and Nox2-deficient mice. Both doses of angiotensin II increased superoxide levels and significantly reduced external diameter in maximally dilated cerebral arterioles in wild-type mice. Increased superoxide and inward remodeling were prevented in Nox2-deficient mice. Moreover, only the pressor dose of AngII increased cross-sectional area of arteriolar wall in wild-type mice and was prevented in Nox2-deficient mice. In conclusion, superoxide derived from Nox2-containing NADPH oxidase plays an important role in angiotensin II-mediated inward remodeling in cerebral arterioles. This effect appears to be independent of pressure and different from that of hypertrophy.

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

Science.gov (United States)

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

2017-04-01

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

β-catenin is selectively required for the expansion and regeneration of mature pancreatic acinar cells in mice

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Matthew D. Keefe

2012-07-01

The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt–β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt–β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

Voltage-gated proton channel is expressed on phagosomes

International Nuclear Information System (INIS)

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

2009-01-01

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

Long-term treatment with the sodium glucose cotransporter 2 inhibitor, dapagliflozin, ameliorates glucose homeostasis and diabetic nephropathy in db/db mice.

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Naoto Terami

Full Text Available Inhibition of sodium glucose cotransporter 2 (SGLT2 has been reported as a new therapeutic strategy for treating diabetes. However, the effect of SGLT2 inhibitors on the kidney is unknown. In addition, whether SGLT2 inhibitors have an anti-inflammatory or antioxidative stress effect is still unclear. In this study, to resolve these issues, we evaluated the effects of the SGLT2 inhibitor, dapagliflozin, using a mouse model of type 2 diabetes and cultured proximal tubular epithelial (mProx24 cells. Male db/db mice were administered 0.1 or 1.0 mg/kg of dapagliflozin for 12 weeks. Body weight, blood pressure, blood glucose, hemoglobin A1c, albuminuria and creatinine clearance were measured. Mesangial matrix accumulation and interstitial fibrosis in the kidney and pancreatic β-cell mass were evaluated by histological analysis. Furthermore, gene expression of inflammatory mediators, such as osteopontin, monocyte chemoattractant protein-1 and transforming growth factor-β, was evaluated by quantitative reverse transcriptase-PCR. In addition, oxidative stress was evaluated by dihydroethidium and NADPH oxidase 4 staining. Administration of 0.1 or 1.0 mg/kg of dapagliflozin ameliorated hyperglycemia, β-cell damage and albuminuria in db/db mice. Serum creatinine, creatinine clearance and blood pressure were not affected by administration of dapagliflozin, but glomerular mesangial expansion and interstitial fibrosis were suppressed in a dose-dependent manner. Dapagliflozin treatment markedly decreased macrophage infiltration and the gene expression of inflammation and oxidative stress in the kidney of db/db mice. Moreover, dapagliflozin suppressed the high-glucose-induced gene expression of inflammatory cytokines and oxidative stress in cultured mProx24 cells. These data suggest that dapagliflozin ameliorates diabetic nephropathy by improving hyperglycemia along with inhibiting inflammation and oxidative stress.

Characteristics of DTH suppressor cells in mice infected with Candida albicans.

Science.gov (United States)

Valdez, J C; Mesón, O E; Sirena, A; de Alderete, N G

1987-05-01

Inoculation of 10(8) C. albicans intraperitoneally into Balb/c mice at given dosage was reported to induce suppression of antigen-specific delayed-type hypersensitivity. Adoptive transfer of spleen cells into normal syngeneic mice pre-treated with Cyclophosphamide confirmed the existence of suppressor cells in mice. Such cells were sensitive to treatment with anti-theta serum and complement, non-adherent to Sephadex G-10. A pretreatment of the mice with Cyclophosphamide eliminated DTH suppression. Treatment with antimacrophage agents via intraperitoneal abrogated suppression only if being effected before inoculation of alive 10(8) Candida albicans. It is concluded that the spleen suppressor cell is a T-lymphocyte whose precursor is Cyclophosphamide-sensitive, requiring the macrophage to be induced.

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-09-15

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

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

Science.gov (United States)

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

2014-09-01

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

Electron-Muon Ranger: performance in the MICE Muon Beam

CERN Document Server

Adams, D.; Vankova-Kirilova, G.; Bertoni, R.; Bonesini, M.; Chignoli, F.; Mazza, R.; Palladino, V.; de Bari, A.; Cecchet, G.; Capponi, M.; Iaciofano, A.; Orestano, D.; Pastore, F.; Tortora, L.; Kuno, Y.; Sakamoto, H.; Ishimoto, S.; Filthaut, F.; Hansen, O.M.; Ramberger, S.; Vretenar, M.; Asfandiyarov, R.; Bene, P.; Blondel, A.; Cadoux, F.; Debieux, S.; Drielsma, F.; Graulich, J.S.; Husi, C.; Karadzhov, Y.; Masciocchi, F.; Nicola, L.; Messomo, E.Noah; Rothenfusser, K.; Sandstrom, R.; Wisting, H.; Charnley, G.; Collomb, N.; Gallagher, A.; Grant, A.; Griffiths, S.; Hartnett, T.; Martlew, B.; Moss, A.; Muir, A.; Mullacrane, I.; Oates, A.; Owens, P.; Stokes, G.; Warburton, P.; White, C.; Adams, D.; Barclay, P.; Bayliss, V.; Bradshaw, T.W.; Courthold, M.; Francis, V.; Fry, L.; Hayler, T.; Hills, M.; Lintern, A.; Macwaters, C.; Nichols, A.; Preece, R.; Ricciardi, S.; Rogers, C.; Stanley, T.; Tarrant, J.; Watson, S.; Wilson, A.; Bayes, R.; Nugent, J.C.; Soler, F.J.P.; Cooke, P.; Gamet, R.; Alekou, A.; Apollonio, M.; Barber, G.; Colling, D.; Dobbs, A.; Dornan, P.; Hunt, C.; Lagrange, J-B.; Long, K.; Martyniak, J.; Middleton, S.; Pasternak, J.; Santos, E.; Savidge, T.; Uchida, M.A.; Blackmore, V.J.; Carlisle, T.; Cobb, J.H.; Lau, W.; Rayner, M.A.; Tunnell, C.D.; Booth, C.N.; Hodgson, P.; Langlands, J.; Nicholson, R.; Overton, E.; Robinson, M.; Smith, P.J.; Dick, A.; Ronald, K.; Speirs, D.; Whyte, C.G.; Young, A.; Boyd, S.; Franchini, P.; Greis, J.; Pidcott, C.; Taylor, I.; Gardener, R.; Kyberd, P.; Littlefield, M.; Nebrensky, J.J.; Bross, A.D.; Fitzpatrick, T.; Leonova, M.; Moretti, A.; Neuffer, D.; Popovic, M.; Rubinov, P.; Rucinski, R.; Roberts, T.J.; Bowring, D.; DeMello, A.; Gourlay, S.; Li, D.; Prestemon, S.; Virostek, S.; Zisman, M.; Hanlet, P.; Kafka, G.; Kaplan, D.M.; Rajaram, D.; Snopok, P.; Torun, Y.; Blot, S.; Kim, Y.K.; Bravar, U.; Onel, Y.; Cremaldi, L.M.; Hart, T.L.; Luo, T.; Sanders, D.A.; Summers, D.J.; Cline, D.; Yang, X.; Coney, L.; Hanson, G.G.; Heidt, C.

2015-12-16

The Muon Ionization Cooling Experiment (MICE) will perform a detailed study of ionization cooling to evaluate the feasibility of the technique. To carry out this program, MICE requires an efficient particle-identification (PID) system to identify muons. The Electron-Muon Ranger (EMR) is a fully-active tracking-calorimeter that forms part of the PID system and tags muons that traverse the cooling channel without decaying. The detector is capable of identifying electrons with an efficiency of 98.6%, providing a purity for the MICE beam that exceeds 99.8%. The EMR also proved to be a powerful tool for the reconstruction of muon momenta in the range 100-280 MeV/$c$.

Induction of interferon by levamisole in mice. [X radiation

Energy Technology Data Exchange (ETDEWEB)

Matsubara, S.; Suzuki, F.; Ishida, N.

1979-03-01

Viral inhibitor(s) with the properties of interferon (IF) was found in the sera of DDI mice injected intraperitoneally with 5 to 10 mg/kg of levamisole. A significant level of IF activity appeared by 20 hr and reached a peak by 24 hr after the injection. The induction was abrogated when the mice were pretreated with either whole-body x irradiation of more than 500 R or 2.5 mg of hydrocortisone acetate but was not affected by macrophage-specific depressors such as carrageenan and trypan blue. Also, no induction was detected in thymus-defective nude mice. These results suggest that thymus-derived lymphocytes in the mouse may be required for IF induction by levamisole.

Electron-muon ranger: performance in the MICE muon beam

International Nuclear Information System (INIS)

Adams, D.; Barclay, P.; Bayliss, V.; Bradshaw, T.W.; Alekou, A.; Apollonio, M.; Barber, G.; Asfandiyarov, R.; Bene, P.; Blondel, A.; De Bari, A.; Bayes, R.; Bertoni, R.; Bonesini, M.; Blackmore, V.J.; Blot, S.; Bogomilov, M.; Booth, C.N.; Bowring, D.; Boyd, S.

2015-01-01

The Muon Ionization Cooling Experiment (MICE) will perform a detailed study of ionization cooling to evaluate the feasibility of the technique. To carry out this program, MICE requires an efficient particle-identification (PID) system to identify muons. The Electron-Muon Ranger (EMR) is a fully-active tracking-calorimeter that forms part of the PID system and tags muons that traverse the cooling channel without decaying. The detector is capable of identifying electrons with an efficiency of 98.6%, providing a purity for the MICE beam that exceeds 99.8%. The EMR also proved to be a powerful tool for the reconstruction of muon momenta in the range 100–280 MeV/c

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

Directory of Open Access Journals (Sweden)

Diana Campelo

2017-10-01

Full Text Available NADPH-cytochrome P450 reductase (CPR is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction, a linker (hinge, and a connecting/FAD domain (NADPH oxidation. It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state to an ensemble of open conformations (unlocked state, the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.

Exhaustion of CTL memory and recrudescence of viremia in lymphocytic choriomeningitis virus-infected MHC class II-deficient mice and B cell-deficient mice

DEFF Research Database (Denmark)

Thomsen, Allan Randrup; Johansen, J; Marker, O

1996-01-01

To study the contribution of CD4+ T cells and B cells to antiviral immunity and long term virus control, MHC class II-deficient and B cell-deficient mice were infected with lymphocytic choriomeningitis virus. In class II-deficient mice, which lack CD4+ T cells, the primary CTL response is virtually...... this phenomenon could reflect participation of B cells and/or Abs in long term virus control, similar experiments were performed with mice that do not have mature B cells because of a disrupted membrane exon of the mu chain gene. In these mice, the cell-mediated immune response was slightly delayed, but transient...... and that in their absence, the virus-specific CTL potential becomes exhausted. Together our results indicate that while CD8+ cells play a dominant role in acute virus control, all three major components of the immune system are required for long term virus control....

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3 radical

International Nuclear Information System (INIS)

Srivastava, S.P.; Chen, N.Q.; Holtzman, J.L.

1990-01-01

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3 radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals and are toxic to cultured hepatocytes. The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4

12/15-lipoxygenase is required for the early onset of high fat diet-induced adipose tissue inflammation and insulin resistance in mice.

Directory of Open Access Journals (Sweden)

Dorothy D Sears

2009-09-01

Full Text Available Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD-induced insulin resistance.Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT and 12/15LO knockout (KO mice after 2-4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b(+, F4/80(+ macrophages and elevated protein levels of the inflammatory markers IL-1beta, IL-6, IL-10, IL-12, IFNgamma, Cxcl1 and TNFalpha. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice.These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.

Time course investigation of PPARα- and Kupffer cell-dependent effects of WY-14,643 in mouse liver using microarray gene expression

International Nuclear Information System (INIS)

Woods, Courtney G.; Kosyk, Oksana; Bradford, Blair U.; Ross, Pamela K.; Burns, Amanda M.; Cunningham, Michael L.; Qu Pingping; Ibrahim, Joseph G.; Rusyn, Ivan

2007-01-01

Administration of peroxisome proliferators to rodents causes proliferation of peroxisomes, induction of β-oxidation enzymes, hepatocellular hypertrophy and hyperplasia, with chronic exposure ultimately leading to hepatocellular carcinomas. Many responses associated with peroxisome proliferators are nuclear receptor-mediated events involving peroxisome proliferators-activated receptor alpha (PPARα). A role for nuclear receptor-independent events has also been shown, with evidence of Kupffer cell-mediated free radical production, presumably through NAPDH oxidase, induction of redox-sensitive transcription factors involved in cytokine production and cytokine-mediated cell replication following acute treatment with peroxisome proliferators in rodents. Recent studies have demonstrated, by using p47 phox -null mice which are deficient in NADPH oxidase, that this enzyme is not related to the phenotypic events caused by prolonged administration of peroxisome proliferators. In an effort to determine the timing of the transition from Kupffer cell-to PPARα-dependent modulation of peroxisome proliferator effects, gene expression was assessed in liver from Pparα-null, p47 phox -null and corresponding wild-type mice following treatment with 4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid (WY-14,643) for 8 h, 24 h, 72 h, 1 week or 4 weeks. WY-14,643-induced gene expression in p47 phox -null mouse liver differed substantially from wild-type mice at acute doses and striking differences in baseline expression of immune related genes were evident. Pathway mapping of genes that respond to WY-14,643 in a time- and dose-dependent manner demonstrates suppression of immune response, cell death and signal transduction and promotion of lipid metabolism, cell cycle and DNA repair. Furthermore, these pathways were largely dependent on PPARα, not NADPH oxidase demonstrating a temporal shift in response to peroxisome proliferators. Overall, this study shows that NADPH oxidase

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

Science.gov (United States)

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

2016-11-01

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

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Ruoting Ding

2016-12-01

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

Cytosolic malic enzyme 1 (ME1 mediates high fat diet-induced adiposity, endocrine profile, and gastrointestinal tract proliferation-associated biomarkers in male mice.

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Ahmed Al-Dwairi

Full Text Available Obesity and associated hormonal disturbances are risk factors for colon cancer. Cytosolic Malic Enzyme (ME1 generates NADPH used for lipogenesis in gastrointestinal (GI, liver and adipose tissues. We have reported that inclusion of soy protein isolate (SPI in the diet lowered body fat content and colon tumor incidence of rats fed AIN-93G diet, while others have demonstrated SPI inhibition of rat hepatic ME1 expression. The present study examined the individual and combined effects of dietary SPI and absence of ME1 on: 1 serum concentrations of hormones implicated in colon cancer development, 2 expression of lipogenic and proliferation-associated genes in the mouse colon and small intestine, and 3 liver and adipose expression of lipogenic and adipocytokine genes that may contribute to colon cancer predisposition.Weanling wild type (WT and ME1 null (MOD-1 male mice were fed high-fat (HF, iso-caloric diets containing either casein (CAS or SPI as sole protein source for 5 wks. Somatic growth, serum hormone and glucose levels, liver and adipose tissue weights, GI tissue parameters, and gene expression were evaluated.The MOD-1 genotype and SPI-HF diet resulted in decreases in: body and retroperitoneal fat weights, serum insulin, serum leptin, leptin/adiponectin ratio, adipocyte size, colon mTOR and cyclin D1 mRNA abundance, and jejunum FASN mRNA abundance, when compared to WT mice fed CAS-HF. Regardless of diet, MOD-1 mice had reductions in liver weight, liver steatosis, and colon crypt depth, and increases in adipose tissue expression of IRS1 and IRS2, compared to WT mice. SPI-HF diet reduced ME1 gene expression only in retroperitoneal fat.Data suggest that the pharmacological targeting of ME1 or the inclusion of soy protein in the diet may provide avenues to reduce obesity and its associated pro-tumorigenic endocrine environment and improve insulin sensitivity, potentially disrupting the obesity-colon cancer connection.

Prodigiosin inhibits gp91{sup phox} and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia-ischemia

Energy Technology Data Exchange (ETDEWEB)

Chang, Chia-Che [Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan (China); Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan (China); Wang, Yea-Hwey [Department of Nursing, College of Medicine and Nursing, Hungkuang University, Taichung, Taiwan (China); Chern, Chang-Ming [Division of Neurovascular Disease, Neurological Institute, Taipei Veterans General Hospital and School of Medicine, National Yang-Ming University, Taipei, Taiwan (China); Liou, Kuo-Tong [Department of Chinese Martial Arts, Chinese Culture University, Taipei, Taiwan (China); Hou, Yu-Chang [Department of Chinese Medicine, Taoyuan General Hospital, Department of Health, Taiwan (China); Department of Nursing, Yuanpei University, Hsinchu, Taiwan (China); Department of Bioscience Technology, Chuan-Yuan Christian University, Taoyuan, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang, E-mail: yuhcs@nricm.edu.tw [National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan (China)

2011-11-15

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100 {mu}g/kg, i.v.) at 1 h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91{sup phox}), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-{kappa}B). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91{sup phox} and iNOS via activation of the NF-{kappa}B pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91{sup phox} and iNOS expression possibly by impairing NF-{kappa}B activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice. -- Highlights: Black-Right-Pointing-Pointer Prodigiosin ameliorated brain infarction and deficits. Black-Right-Pointing-Pointer Prodigiosin protected against hypoxia/reperfusion-induced brain injury. Black-Right-Pointing-Pointer Prodigiosin diminished oxidative/nitrosativestress and leukocytes infiltration. Black-Right-Pointing-Pointer Prodigiosin reduced BBB breakdown. Black

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

Science.gov (United States)

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice

International Nuclear Information System (INIS)

Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna; Chowdhury, Abhijit; Boyer, James L.; Santra, Amal

2011-01-01

Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 μg/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-β1, PDGF-Rβ, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro(α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.

Evolution of Enzymes Required for Biodegradation of Pentachlorophenol in Sphingobium Chlorophenolicum

National Research Council Canada - National Science Library

Copley, Shelley D

2007-01-01

.... Furthermore, it catalyzes a futile cycling reaction in which C4a-hydroperoxyflavin at the active site decays to produce H2O2 without hydroxylating the substrate, thus wasting NADPH and producing a toxic by-product...

Lipid-derived free radical production in superantigen-induced interstitial pneumonia

Science.gov (United States)

Miyakawa, Hisako; Mason, Ronald P.; Jiang, JinJie; Kadiiska, Maria B.

2009-01-01

We studied the free radical generation involved in the development of interstitial pneumonia (IP) in an animal model of autoimmune disease. We observed an electron spin resonance (ESR) spectrum of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts detected in the lipid extract of lungs in autoimmune-prone mice after intratracheal instillation of staphylococcal enterotoxin B. The POBN adducts detected by ESR were paralleled by infiltration of macrophages and neutrophils in the bronchoalveolar lavage fluid. To further investigate the mechanism of free radical generation, mice were pretreated with the macrophage toxicant gadolinium chloride, which significantly suppressed the radical generation. Free radical generation was also decreased by pretreatment with the xanthine oxidase (XO) inhibitor allopurinol, the iron chelator Desferal, and the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Histopathologically, these drugs significantly reduced both the cell infiltration to alveolar septal walls and the synthesis of pulmonary collagen fibers. Experiments with NADPH oxidase knockout mice showed that NADPH oxidase did not contribute to lipid radical generation. These results suggest that lipid-derived carbon-centered free radical production is important in the manifestation of IP and that a macrophage toxicant, an XO inhibitor, an iron chelator, and an iNOS inhibitor protect against both radical generation and the manifestation of IP. PMID:19376221

Life cycle biological efficiency of mice divergently selected for heat loss.

Science.gov (United States)

Bhatnagar, A S; Nielsen, M K

2014-08-01

Divergent selection in mice for heat loss was conducted in 3 independent replicates creating a high maintenance, high heat loss (MH) and low maintenance, low heat loss (ML) line and unselected control (MC). Improvement in feed efficiency was observed in ML mice due to a reduced maintenance energy requirement but there was also a slight decline in reproductive performance, survivability, and lean content, particularly when compared to MC animals. The objective of this study was to model a life cycle scenario similar to a livestock production system and calculate total inputs and outputs to estimate overall biological efficiency of these lines and determine if reduced feed intake resulted in improved life cycle efficiency. Feed intake, reproductive performance, growth, and body composition were recorded on 21 mating pairs from each line × replicate combination, cohabitated at 7 wk of age and maintained for up to 1 yr unless culled. Proportion of animals at each parity was calculated from survival rates estimated from previous research when enforcing a maximum of 4, 8, or 12 allowed parities. This parity distribution was then combined with values from previous studies to calculate inputs and outputs of mating pairs and offspring produced in a single cycle at equilibrium. Offspring output was defined as kilograms of lean output of offspring at 49 d. Offspring input was defined as megacalories of energy intake for growing offspring from 21 to 49 d. Parent output was defined as kilograms of lean output of culled parents. Parent input was defined as megacalories of energy intake for mating pairs from weaning of one parity to weaning of the next. Offspring output was greatest in MC mice due to superior BW and numbers weaned, while output was lowest in ML mice due to smaller litter sizes and lean content. Parent output did not differ substantially between lines but was greatest in MH mice due to poorer survival rates resulting in more culled animals. Input was greatest in

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity

Science.gov (United States)

Intini, Giuseppe; Nyman, Jeffry S.

2015-01-01

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures has been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1+/−) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2c/c;Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1+/− mice were crossed with Bmp2c/c;Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy. PMID:25603465

Impaired memory of eyeblink conditioning in CaMKIV KO mice.

Science.gov (United States)

Lee, Ka Hung; Chatila, Talal A; Ram, Rana A; Thompson, Richard F

2009-04-01

The calcium/calmodulin-dependent protein kinase type IV (CaMKIV) is highly expressed in cerebellar cortical granule cells and deep nuclear neurons in the cerebellum. It mediates the phosphorylation and activation of the cAMP-dependent response element binding protein (CREB). In several paradigms CREB-dependent transcription is required for cellular events underlying long-term memory processes. Also, CaMKIV deficiency results in impaired long-term depression (LTD) induction in cerebellar cortex. To investigate the function of CaMKIV in the cerebellum, Wild-type (WT) and CaMKIV KO mice were tested with delay eyeblink conditioning. KO and WT mice did not differ in acquisition, but the KO mice showed a significantly lower conditioned response (CR) percentage than the WT mice in the retention testing and retraining period. The CR peak latencies for the two groups did not differ in acquisition but were shorter for the KO mice in the testing period. No significant differences were found between KO and WT mice in spontaneous eyeblink activity, auditory brainstem response (ABR) amplitudes, and tail-flick latency. The results suggest an important role for CaMKIV in long-term memory in the cerebellum. (c) 2009 APA, all rights reserved.

Psoralea corylifolia L. Attenuates Nonalcoholic Steatohepatitis in Juvenile Mouse

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Lishan Zhou

2017-11-01

Full Text Available Psoralea corylifolia L. (PC is a traditional Chinese herb used to treat yang deficiency of the spleen and kidney in pediatric disease. Recent studies have shown its liver protection and anti-oxidative effects. The aim of this study was to explore the effect and mechanism of PC on nonalcoholic steatohepatitis in juvenile mice. The juvenile mouse model of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH was established by being fed a high-fat diet in maternal-offspring manner. PC granules were prepared and the quality was assessed. The main components were identified by high performance liquid chromatography. Then, different dosages of PC were administered for 6 weeks. Homeostatic model assessment of insulin resistance, plasma liver enzymes, hepatic morphology, hepatic superoxide anion, and triglyceride/total cholesterol levels were examined. The changes of nuclear factor-κB (NF-κB activity phosphatidylinositol 3 kinase (PI3K/protein kinase B (Akt and protein kinase C-α (PKC-α/nicotinamide-adenine dinucleotide phosphate (NADPH oxidase signaling pathways in hepatic tissues were also determined. Our data demonstrated that PC significantly improved liver dysfunction, liver triglyceride/total cholesterol accumulation and insulin resistance in juvenile NAFLD/NASH mice. PC also alleviated hepatic steatosis, inflammatory cell infiltration, and fibroplasia in the portal area. Additionally, PC inhibited the activation of NF-κB and the mRNA expression of inflammatory factors while enhancing PI3K/Akt signaling in hepatic tissues. PC could also reduce hepatic superoxide anion levels, and NADPH oxidase activity as well as p47phox protein expression and PKCα activation in hepatic tissues. The results suggest that PC is effective in the treatment of NASH in juvenile mice. The mechanism may be related to the attenuation of hepatic oxidative stress through the PKC-α/NADPH oxidase signaling pathway.

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-05-27

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

Sesamin Ameliorates Advanced Glycation End Products-Induced Pancreatic β-Cell Dysfunction and Apoptosis

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Xiang Kong

2015-06-01

Full Text Available Advanced glycation end products (AGEs, the direct modulators of β-cells, have been shown to cause insulin-producing β-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked β-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg and orally treated with sesamin (160 mg/kg for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and β-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 μM and then exposed to AGEs (200 mg/L for 24 h. Insulin secretion, β-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced β-cell dysfunction and apoptosis both in vivo and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67phox and p22phox, and reduced NADPH oxidase activity. These results suggest that sesamin protects β-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.

Spatial reversal learning in preclinical scrapie-inoculated mice.

Science.gov (United States)

Lysons, A M; Woollard, S J

1996-04-10

Acquisition and reversal of a two-choice spatial discrimination were tested in scrapie-inoculated mice. Both acquisition and reversal were normal in mice tested 138 and 103 days prior to the onset of clinical symptoms. At 65 days before onset of clinical symptoms, scrapie-inoculated mice required more trails to criterion in reversal learning, but this effect was not significant in a second experiment (68 days preclinical) and was transient: no effect was seen 33 days before symptoms. However, the course of reversal learning was abnormal in all three late preclinical groups (68, 65 and 33 days before symptoms). Reversal learning in these three groups was characterized by a rapid extinction of the original discrimination, followed by a period, absent in controls, during which performance showed no further improvement. This effect corresponds in time of onset to the appearance of characteristic neuropathological features.

PGC-1alpha mediates exercise-induced skeletal muscle VEGF expression in mice

DEFF Research Database (Denmark)

Leick, Lotte; Hellsten, Ylva; Fentz, Joachim

2009-01-01

The aim of the present study was to test the hypothesis that PGC-1alpha is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1alpha-dependent mechanism. Whole body PGC-1alpha knockout (KO......) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1alpha KO mice, VEGF protein...... expression was approximately 60-80% lower and the capillary-to-fiber ratio approximately 20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1alpha KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased...

Comparative studies between mice molars and incisors are required to draw an overview of enamel structural complexity

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MICHEL eGOLDBERG

2014-09-01

Full Text Available In the field of dentistry, the murine incisor has long been considered as an outstanding model to study amelogenesis. However, it clearly appears that enamel from wild type mouse incisors and molars presents several structural differences. In incisor, exclusively radial enamel is observed. In molars, enamel displays a high level of complexity since the inner part is lamellar whereas the outer enamel shows radial and tangential structures. Recently, the serotonin 2B receptor (5-HT2BR was shown to be involved in ameloblast function and enamel mineralization. The incisors from 5HT2BR knockout (KO mice exhibit mineralization defects mostly in the outer maturation zone and porous matrix network in the inner zone. In the molars, the mutation affects both secretory and maturation stages of amelogenesis since pronounced alterations concern overall enamel structures. Molars from 5HT2BR KO mice display reduction in enamel thickness, alterations of inner enamel architecture including defects in Hunter-Schreger Bands arrangements, and altered maturation of the outer radial enamel. Differences of enamel structure were also observed between incisor and molar from other KO mice depleted for genes encoding enamel extracellular matrix proteins.

Correlation between Mitochondrial Reactive Oxygen and Severity of Atherosclerosis

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Gabriel G. Dorighello

2016-01-01

Full Text Available Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

Science.gov (United States)

Cotter, David G; Schugar, Rebecca C; Wentz, Anna E; d'Avignon, D André; Crawford, Peter A

2013-02-15

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

Antibody response is required for protection from Theiler's virus-induced encephalitis in C57BL/6 mice in the absence of CD8+ T cells

International Nuclear Information System (INIS)

Kang, B.-S.; Palma, Joann P.; Lyman, Michael A.; Dal Canto, Mauro; Kim, Byung S.

2005-01-01

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that β 2 M-deficient C57BL/6 mice lacking functional CD8 + T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice (μMT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected μMT and control B6 mice, the levels of CD4 + T cells were higher in the CNS of μMT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in μMT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated μMT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8 + T cells

Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots.

Science.gov (United States)

Ruíz-Torres, Carmelo; Feriche-Linares, Rafael; Rodríguez-Ruíz, Marta; Palma, José M; Corpas, Francisco J

2017-04-01

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200μM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

Science.gov (United States)

Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

2017-04-01

The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

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

Science.gov (United States)

Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

2017-07-01

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

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

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Parhad, Swapnil S; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

2016-03-25

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. Copyright © 2016 Elsevier Inc. All rights reserved.

MyD88 Adaptor Protein Is Required for Appropriate Hepcidin Induction in Response to Dietary Iron Overload in Mice

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Antonio Layoun

2018-03-01

Full Text Available Iron homeostasis is tightly regulated to provide virtually all cells in the body, particularly red blood cells, with this essential element while defending against its toxicity. The peptide hormone hepcidin is central to the control of the amount of iron absorbed from the diet and iron recycling from macrophages. Previously, we have shown that hepcidin induction in macrophages following Toll-like receptor (TLR stimulation depends on the presence of myeloid differentiation primary response gene 88 (MyD88. In this study, we analyzed the regulation of iron metabolism in MyD88−/− mice to further investigate MyD88 involvement in iron sensing and hepcidin induction. We show that mice lacking MyD88 accumulate significantly more iron in their livers than wild-type counterparts in response to dietary iron loading as they are unable to appropriately control hepcidin levels. The defect was associated with inappropriately low levels of Smad4 protein and Smad1/5/8 phosphorylation in liver samples found in the MyD88−/− mice compared to wild-type mice. In conclusion, our results reveal a previously unknown link between MyD88 and iron homeostasis, and provide new insights into the regulation of hepcidin through the iron-sensing pathway.

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

Science.gov (United States)

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

2015-03-28

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

Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

Science.gov (United States)

Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

2014-11-01

The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

Neuronal nitric oxide synthase-rescue of dystrophin/utrophin double knockout mice does not require nNOS localization to the cell membrane.

Directory of Open Access Journals (Sweden)

Michelle Wehling-Henricks

Full Text Available Survival of dystrophin/utrophin double-knockout (dko mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS transgene. Dko mice expressing the transgene (nNOS TG+/dko experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span.

Global inhibition of DC priming capacity in the spleen of self-antigen vaccinated mice requires IL-10

Directory of Open Access Journals (Sweden)

Douglas Matthew Marvel

2014-02-01

Full Text Available DC in the spleen are highly activated following intravenous vaccination with a foreign antigen, promoting expansion of effector T cells, but remain phenotypically and functionally immature after vaccination with a self-antigen. Up-regulation or suppression of expression of a cohort of pancreatic enzymes 24-72 hours post-vaccination can be used as a biomarker of stimulatory versus toleragenic DC, respectively. Here we show, using MUC1 transgenic mice (MUC1.Tg and a vaccine based on the MUC1 peptide which these mice perceive as a self-antigen, that the difference in enzyme expression that predicts whether DC will promote immune response or immune tolerance, is seen as early as 4-8 hours following vaccination. We also identify early production of IL-10 as a predominant factor that both correlates with this early time point and controls DC function. Pre-treating mice with an antibody against the IL-10 receptor (IL-10R prior to vaccination results in DC that up-regulate CD40, CD80, and CD86 and promote stronger IFNγ+ T cell responses. This study suggests that transient inhibition of IL-10 prior to vaccination could improve responses to cancer vaccines that utilize self-tumor antigens.

Characterization of the insulin sensitivity of ghrelin receptor KO mice using glycemic clamps

Directory of Open Access Journals (Sweden)

Morgan Kristen

2011-01-01

Full Text Available Abstract Background We and others have demonstrated previously that ghrelin receptor (GhrR knock out (KO mice fed a high fat diet (HFD have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG and hyperinsulinemic-euglycemic (HI-E clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity. Results Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd, and decreased hepatic glucose production (HGP. HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice. Conclusions These results indicate that improved glucose homeostasis of GhrR KO mice is

Impact of food restriction and cocaine on locomotion in ghrelin- and ghrelin-receptor knockout mice.

Science.gov (United States)

Clifford, Shane; Zeckler, Rosie Albarran; Buckman, Sam; Thompson, Jeff; Hart, Nigel; Wellman, Paul J; Smith, Roy G

2011-07-01

Food restriction (FR) augments the behavioral and reinforcing effects of psychomotor stimulants such as cocaine or amphetamine; effects that may be related to the capacity of FR to increase plasma levels of ghrelin (GHR), a 28-amino acid orexigenenic peptide linked to activation of brain dopamine systems. The present study used wild-type (WT) mice or mutant mice sustaining knockout of either GHR [GHR((-/-)) ] or of the growth hormone secretagogue receptor [GHS-R((-/-)) ] and subjected to FR or not to evaluate the role of GHR and GHS-R in cocaine-stimulated locomotion. WT, GHR((-/-)) , and GHS-R((-/-)) mice were either restricted to 60% of baseline caloric intake or allowed to free-feed (FF). Mice were treated with 0, 1.25, 2.5 and 5.0 mg/kg cocaine on separate test days (in random dose order) and forward locomotion was recorded on each drug day for 45 minutes after drug dosing. Food (and water) was available immediately after (but not during) each activity test. For FF mice, there was no interaction between cocaine and GHR status on locomotion. FR-WT mice treated with saline exhibited significant increases in anticipatory locomotion (relative to FF-WT mice), whereas FR-GHS-R((-/-)) mice did not. Cocaine significantly increased locomotion in FR-GHR((-/-)) and FR-GHS-R((-/-)) mice to the levels noted in FR-WT mice. These results suggest that GHS-R activity, but not GHR activity, is required for FR to augment food-associated anticipatory locomotion, but do not support the contention that GHR pathways are required for the capacity of FR to augment the acute effect of cocaine on locomotion. © 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction.