Exogenous NADPH Research Articles

Detection of NAD(P)H by fluorescent probe to reveal the protective effects of natural antioxidants on acute liver injury

Acute liver injury as a serious inflammatory liver disease has a high morbidity and mortality rate. For drug treatment for acute liver injury, the focus was mainly from oxidative stress and rarely from a reductive perspective over the past decades. Herein, a novel quinoline-malononitrile-based fluorescent probe (QM-NA) designed for high efficiency sensitive detection of NAD(P)H, a critical marker related to reductive stress. QM-NA exhibited high selectivity for NADPH as well as a fast "turn-on" fluorescence response (4 min) with a Stokes shift of 77 nm, a detection limit of 18 nM, and favorable biocompatibility for cell-based experiments. With QM-NA in hand, the fluorescence imaging of endogenous and exogenous NADPH at the cellular level was achieved. More importantly, in vivo experiments demonstrated QM-NA was successfully used to visualize the fluctuations of NADPH and evaluate the efficacy of three antioxidants for the first time in acute liver injury mice. These above results afford a novel strategy for the treatment of acute liver injury and this probe is expected to be a promising molecular tool to evaluate the efficacy of hepatoprotective drugs.

Interaction Between the PERK/ATF4 Branch of the Endoplasmic Reticulum Stress and Mitochondrial One-Carbon Metabolism Regulates Neuronal Survival After Intracerebral Hemorrhage.

Recent investigations have revealed that oxidative stress can lead to neuronal damage and disrupt mitochondrial and endoplasmic reticulum functions after intracerebral hemorrhage (ICH). However, there is limited evidence elucidating their role in maintaining neuronal homeostasis. Metabolomics analysis, RNA sequencing, and CUT&Tag-seq were performed to investigate the mechanism underlying the interaction between the PERK/ATF4 branch of the endoplasmic reticulum stress (ERS) and mitochondrial one-carbon (1C) metabolism during neuronal resistance to oxidative stress. The association between mitochondrial 1C metabolism and the PERK/ATF4 branch of the ERS after ICH was investigated using transcription factor motif analysis and co-immunoprecipitation. The findings revealed interactions between the GRP78/PERK/ATF4 and mitochondrial 1C metabolism, which are important in preserving neuronal homeostasis after ICH. ATF4 is an upstream transcription factor that directly regulates the expression of 1C metabolism genes. Additionally, the GRP78/PERK/ATF4 forms a negative regulatory loop with MTHFD2 because of the interaction between GRP78 and MTHFD2. This study presents evidence of disrupted 1C metabolism and the occurrence of ERS in neurons post-ICH. Supplementing exogenous NADPH or interfering with the PERK/ATF4 could reduce symptoms related to neuronal injuries, suggesting new therapeutic prospects for ICH.

Exogenous NADPH exerts a positive inotropic effect and enhances energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure

Therapies are urgently required to ameliorate pathological cardiac hypertrophy and enhance cardiac function in heart failure. Our preliminary experiments have demonstrated that exogenous NADPH exhibits a positive inotropic effect on isolated heart. This study aims to investigate the positive inotropic effects of NADPH in pathological cardiac hypertrophy and heart failure, as well as the underlying mechanisms involved. Endogenous plasma NADPH contents were determined in patients with chronic heart failure and control adults. The positive inotropic effects of NADPH were investigated in isolated toad heart or rat heart. The effects of NADPH were investigated in isoproterenol (ISO)-induced cardiac hypertrophy or transverse aortic constriction (TAC)-induced heart failure. The underlying mechanisms of NADPH were studied using SIRT3 knockout mice, echocardiography, Western blotting, transmission electron microscopy, and immunoprecipitation. The endogenous NADPH content in the blood of patients and animals with pathological cardiac hypertrophy or heart failure was significantly reduced compared with age-sex matched control subjects. Exogenous NADPH showed positive inotropic effects on the isolated normal and failing hearts, while antagonism of ATP receptor partially abolished the positive inotropic effect of NADPH. Exogenous NADPH administration significantly reduced heart weight indices, and improved cardiac function in the mice with pathological cardiac hypertrophy or heart failure. NADPH increased SIRT3 expression and activity, deacetylated target proteins, improved mitochondrial function and facilitated ATP production in the hypertrophic myocardium. Importantly, inhibition of SIRT3 abolished the positive inotropic effect of NADPH, and the anti-heart failure effect of NADPH was significantly reduced in the SIRT3 Knockout mice. Exogenous NADPH shows positive inotropic effect and improves energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure. NADPH thus may be one of the potential candidates for the treatment of pathological cardiac hypertrophy or heart failure. This work was supported by grants from the National Natural Science Foundation of China (No. 81973315, 82173811, 81730092), Natural Science Foundation of Jiangsu Higher Education (20KJA310008), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

Ca2+-dependent oxidation of exogenous NADH and NADPH by the mitochondria of spring wheat and its relation with AOX capacity and ROS content at high temperatures

Mitochondria are sources of reactive oxygen species (ROS) in a plant cell under high temperature. Mitochondrial alternative NAD(P)H dehydrogenases (type II NAD(P)H DHs) and cyanide-resistant oxidase (AOX) can regulate ROS production, but their role at high temperatures is unknown. This study investigates the influence heat acclimation (37 °C) and heat shock (50 °C) temperatures on ROS content, activity and protein abundance of external Ca2+-dependent NAD(P)H DHs (NDB) and AOX in mitochondria of 4- and 8-day-old seedlings of spring wheat (Triticum aestivum L., var. Novosibirskya 29). The shoots of 4-day-old seedlings contained more carbohydrates, had a higher rate of total respiration and a high rate of oxidation of exogenous NADH, a greater AOX capacity and a lower of ROS content, as compared to leaves of 8-day-old seedlings, and were more resistant to heat shock. The activity of external NADH DH was higher than the one of NADPH DH in mitochondria of both shoots and leaves. At 37 °C, high NADH oxidation was associated with increased AOX capacity in mitochondria of both shoots and leaves, whereas NADPH oxidation with COX capacity. At 50 °C, the NADPH oxidation by shoots’ mitochondria increased and the NADH oxidation stayed high. The content of NDB and AOX proteins depends on heat treatments and differs between mitochondria of shoots and leaves. Our data indicate that Ca2+-dependent type II NAD(P)H DHs can regulate the ROS content and together with AOX are involved in heat tolerance, depending on the development phase of spring wheat and is, probably, tissue-specific.

Effects of ROS and caspase-3-like protein on the growth and aerenchyma formation of Potamogeton perfoliatus stem.

Aerenchyma formation plays an important role in the survival of Potamogeton perfoliatus in submerged environment. To understand the regulatory role of reactive oxygen species (ROS) and caspase 3-like protein signaling molecules in aerenchyma formation, we investigated the effects of exogenous NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), catalase inhibitor (3-amino-1,2,4-triazole, AT), and caspase-3-like protein inhibitor (AC-DEVD-CHO, DEVD) on morphological and physiological characteristics and aerenchyma formation in P. perfoliatus. The results showed that after DPI treatment, caspase-3-like protein activity decreased, ROS-related enzyme activities increased, and H2O2 content decreased, thereby inhibiting aerenchyma formation. When the concentration of DPI was approximately 1μmol/L, the inhibitory effect was the most obvious. On the contrary, after the AT treatment, caspase-3-like protein activity increased, ROS-related enzyme activities decreased, and the H2O2 content increased, ultimately promoting aerenchyma formation, and the promotion was the most obvious under treatment with approximately 500μmol/L AT. After DEVD treatment, the inhibition of vegetative growth caused by DPI or AT treatment was alleviated, significantly reducing caspase-3-like activity and inhibiting aerenchyma development. The results of this study show that ROS has a positive regulatory effect on aerenchyma formation, and caspase-3-like protein is activated to promote ROS-mediated aerenchyma formation. This experiment provides a new theoretical basis for further exploration of the signal transduction effects of ROS and caspase-3-like protein in plant cells and their roles in plant development.

NADPH levels affect cellular epigenetic state by inhibiting HDAC3-Ncor complex.

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

A cinnamyl alcohol dehydrogenase required for sclerotial development in Sclerotinia sclerotiorum

Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

Semi-rational engineering of carbonyl reductase YueD for efficient biosynthesis of halogenated alcohols with in situ cofactor regeneration

Optically active 1-phenylethanol and its derivatives are versatile chiral precursors for many pharmaceuticals. The increasing market demand of enantiopure alcohols calls for exploration of more robust biocatalysts capable of delivering high conversion rates at high substrate concentrations. The carbonyl reductase YueD from Bacillus subtilis was engineered for improved reduction of halogenated acetophenones. Based on in silico docking and Alanine screening, mutant Val181Ala with 97% conversion of the model substrate 3-bromoacetophenone was obtained. Further molecular simulation analysis suggested expansion of the catalytic pocket and altered substrate orientation to provide a structural basis for the improved activity upon Val181 mutation. To eliminate the requirement of expensive exogenous NADPH and thus afford economical synthesis of enantiopure alcohol products, a cofactor regeneration system based on glucose dehydrogenase was introduced. Since the product of glucose oxidation seemed to inhibit the activity of YueD, xylose was used instead as the co-substrate, which efficiently regenerated NADPH in situ, delivering (S) or (R) alcohols with >99% ee at high conversion rates of 85–99% in the asymmetric reduction of halogenated acetophenones (500 mM). These data demonstrate the industrial potential of the YueD mutant Val181Ala in biosynthesis of valuable chiral alcohols.

Asymmetric Reduction of Prochiral Ketones by Using Self-Sufficient Heterogeneous Biocatalysts Based on NADPH-Dependent Ketoreductases.

The development of cell-free and self-sufficient biocatalytic systems represents an emerging approach to address more complex synthetic schemes under nonphysiological conditions. Herein, we report the development of a self-sufficient heterogeneous biocatalyst for the synthesis of chiral alcohols without the need to add an exogenous cofactor. In this work, an NADPH-dependent ketoreductase was primarily stabilized and further co-immobilized with NADPH to catalyze asymmetric reductions without the addition of an exogenous cofactor. As a result, the immobilized cofactor is accessible, and thus, it is recycled inside the porous structure without diffusing out into the bulk, as demonstrated by single-particle in operando studies. This self-sufficient heterogeneous biocatalyst was used and recycled for the asymmetric reduction of eleven carbonyl compounds in a batch reactor without the addition of exogenous NADPH to achieve the corresponding alcohols in 100 % yield and >99 % ee; this high performance was maintained over five consecutive reaction cycles. Likewise, the self-sufficient heterogeneous biocatalyst was integrated into a plug flow reactor for the continuous synthesis of one model secondary alcohol, which gave rise to a space-time yield of 97-112 g L-1 day-1 ; additionally, the immobilized cofactor accumulated a total turnover number of 1076 for 120 h. This is one of the few examples of the successful implementation of continuous reactions in aqueous media catalyzed by cell-free and immobilized systems that integrate both enzymes and cofactors into the solid phase.

G6PD plays a neuroprotective role in brain ischemia through promoting pentose phosphate pathway

TIGAR-regulated pentose phosphate pathway (PPP) plays a critical role in the neuronal survival during cerebral ischemia/reperfusion. Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme in PPP and thus, we hypothesized that it plays an essential role in anti-oxidative defense through producing NADPH. The present study investigated the regulation and the role of G6PD in ischemia/reperfusion-induced neuronal injury with in vivo and in vitro models of ischemic stroke. The results showed that the levels of G6PD mRNA and protein were increased after ischemia/reperfusion. In vivo, lentivirus-mediated G6PD overexpression in mice markedly reduced neuronal damage after ischemia/reperfusion insult, while lentivirus-mediated G6PD knockdown exacerbated it. In vitro, overexpression of G6PD in cultured primary neurons decreased neuronal injury under oxygen and glucose deprivation/reoxygenation (OGD/R) condition, whereas knockdown of G6PD aggravated it. Overexpression of G6PD increased levels of NADPH and reduced form of glutathione (rGSH), and ameliorated ROS-induced macromolecular damage. On the contrary, knockdown of G6PD executed the opposite effects in mice and in primary neurons. Supplementation of exogenous NADPH alleviated the detrimental effects of G6PD knockdown, whereas further enhanced the beneficial effects of G6PD overexpression in ischemic injury. Therefore, our results suggest that G6PD protects ischemic brain injury through increasing PPP. Thus G6PD may be considered as potential therapeutic target for treatment of ischemic brain injury.

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.

X‐ROS Signaling in Cerebral Artery Smooth Muscle

Mechanically induced generation of reactive oxygen species (ROS) by NADPH oxidase (NOX), referred to as X‐ROS, influences the contractility of cardiac and skeletal muscle, but the impact of this signaling pathway in smooth muscle has not been reported. Here we investigate the effect of X‐ROS on the excitability and contractility of cerebral artery smooth muscle cells (SMC). In these cells, spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR) generate microdomains of high intracellular [Ca2+] known as Ca2+ sparks. Ca2+ sparks are functionally coupled to multiple large‐conductance Ca2+‐activated K+ (BK) channels and activate macroscopic Spontaneous Transient Outward Currents (STOCs) that cause membrane hyperpolarization and vasodilation. Several sites subject to redox modification are present on RyRs that influence activity. Therefore, we hypothesized that X‐ROS elevates Ca2+ spark and STOC activity, leading to membrane hyperpolarization and vasodilation. Experiments were carried out using cerebral pial arteries and freshly isolated cerebral artery SMC from C57/Bl6 mice. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. To determine if the production of O2− was required for generation of Ca2+ sparks, superoxide dismutase (SOD) was applied to the bath. Membrane permeable PEG‐SOD (100 U.ml−1, n = 5) nearly abolished Ca2+ spark activity. To further characterize this response, SMC were patch‐clamped using the amphotericin B perforated‐patch whole cell method to record STOCs. PEG‐SOD (100 U.ml−1, n = 6) attenuated STOC frequency, whereas non‐membrane permeable SOD (100 U.ml−1, n = 7) had no effect. The membrane permeable SOD mimetic TEMPO (1 mM, n = 5) also, attenuated STOC activity. Catalase (750 U.ml−1, n = 5) or membrane permeable PEG‐catalase (750 U.ml−1, n = 6) had no effect on STOC frequency, suggesting that H2O2 intermediates are not involved in this signaling pathway. Inhibition of NOX2 with apocynin (30 μM, n = 7) or gp91ds‐tat (1 μM, n = 6) greatly attenuated STOC frequency, whereas application of exogenous NADPH to increase NOX2 activity resulted in a concentration‐dependent increase in STOC frequency, with an EC50 of 61 nM (n = 3–8). These data indicate that intracellular generation of O2− by NOX2 is necessary for Ca2+ spark and STOC activity. Pressure myography experiments were performed to determine the functional significance of the X‐ROS pathway in SMC. Endothelium denuded posterior cerebral arteries were pressurized to 50 mmHg to generate spontaneous myogenic tone and then treated with the substrate for NOX, NADPH (10 μM). NADPH caused vasodilation that was reversed by the BK blocker paxilline (1 μM, n = 5), suggesting that NOX activity dilates cerebral arteries by activating BK channels. We conclude that generation of intracellular O2− by NOX2 stimulates Ca2+ spark and STOC activity, resulting in cerebral arteries dilation. These data highlight a novel ROS‐dependent signaling pathway that may critically influence cerebral blood flow regulation.Support or Funding InformationSupport: R01HL091905 (SE), AHA15POST2472002 (PWP).

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates, yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP+ to NADPH by ferredoxin–NADP+-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP++NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.

Astragaloside IV attenuates glycated albumin-induced epithelial-to-mesenchymal transition by inhibiting oxidative stress in renal proximal tubular cells

In diabetic kidney disease (DKD), epithelial-to-mesenchymal transition (EMT) is a classic pathological process in tubular damage. Oxidative stress is considered to play an important role in DKD. Astragaloside IV (A-IV), one of the main active ingredients of Astragalus membranaceus, exhibits a wide range of biological activities. However, the effect of A-IV on regulating EMT in tubular cells is unclear. This study aims to determine whether A-IV could attenuate glycated albumin (GA)-induced EMT in the NRK-52E cell line by inhibiting oxidative stress. GA and A-IV-induced cytotoxicity were assayed by CCK-8. The intercellular reactive oxygen species (ROS) level was detected by H2DCFDA. The activity of NADPH oxidase was assayed by adding exogenous NADPH oxidase, and the superoxide dismutase (SOD) units were observed by NBT. We used a microscope to examine the morphology of the NRK-52E cell line. We conducted a wound healing assay to measure cell mobility. To determine mRNA and protein expressions of α-SMA and E-cadherin, we used real-time polymerase chain reaction (real-time PCR), immunofluorescence, and western blot analysis. A-IV significantly attenuated GA-induced amplification of ROS, lowered the increased level of NADPH oxidase activity, and elevated the decreased level of SOD units. The GA-induced NRK-52E cell line showed increased expression of α-SMA and decreased expression of E-cadherin in mRNA and protein levels, whereas A-IV alleviated the expression of α-SMA and increased the expression of E-cadherin. Our data demonstrate that GA could induce NRK-52E cell line EMT through oxidative stress. This effect could be attenuated by A-IV via regulation of the impaired redox balance.

Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3+ pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.

Characterization of Nda2, a Plastoquinone-reducing Type II NAD(P)H Dehydrogenase in Chlamydomonas Chloroplasts

Electron transfer pathways associated to oxygenic photosynthesis, including cyclic electron flow around photosystem I and chlororespiration, rely on non-photochemical reduction of plastoquinones (PQs). In higher plant chloroplasts, a bacterial-like NDH complex homologous to complex I is involved in PQ reduction, but such a complex is absent from Chlamydomonas plastids where a type II NAD(P)H dehydrogenase activity has been proposed to operate. With the aim to elucidate the nature of the enzyme-supporting non-photochemical reduction of PQs, one of the type II NAD(P)H dehydrogenases identified in the Chlamydomonas reinhardtii genome (Nda2) was produced as a recombinant protein in Escherichia coli and further characterized. As many type II NAD(P)H dehydrogenases, Nda2 uses NADH as a preferential substrate, but in contrast to the eukaryotic enzymes described so far, contains non-covalently bound FMN as a cofactor. When expressed at a low level, Nda2 complements growth of an E. coli lacking both NDH-1 and NDH-2, but is toxic at high expression levels. Using an antibody raised against the recombinant protein and based on its mass spectrometric identification, we show that Nda2 is localized in thylakoid membranes. Chlorophyll fluorescence measurements performed on thylakoid membranes show that Nda2 is able to interact with thylakoid membranes of C. reinhardtii by reducing PQs from exogenous NADH or NADPH. We discuss the possible involvement of Nda2 in cyclic electron flow around PSI, chlororespiration, and hydrogen production.

Visual cycle and its metabolic support in gecko photoreceptors

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all- trans retinal to all- trans retinol, and the clearance of all- trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all- trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP + glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

NADPH‐induced contractions of mouse aorta do not involve NADPH oxidase: A role for P2X receptors

Reactive oxygen species elicit vascular effects ranging from dilatation due to hydrogen peroxide-mediated opening of K+ channels, to contraction arising from superoxide (O2−) -dependent inactivation of endothelium-derived nitric oxide. Given that NADPH oxidases are major sources of O2− in the vascular wall, this study examined the effects of exogenous NADPH, a substrate of these enzymes, on O2− generation (lucigenin) and vascular tone (isometric tension) in mouse isolated aortic rings. NADPH caused concentration-dependent increases in O2− generation and vascular tone. However, while oxidised NADP+ was unable to support O2− production, it was equally as effective as reduced NADPH at stimulating contraction. An NADPH oxidase inhibitor, diphenyleneiodonium, markedly attenuated NADPH-induced O2− production, yet had no effect on contractions to NADPH. In contrast, two P2X receptor antagonists, PPADS and NF023, markedly attenuated both endothelium-dependent and -independent contractions to NADPH, as did the P2X desensitising agent, α,β-methylene-ATP. Importantly, α,β-methylene-ATP had no effect on O2− production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived O2− in the contractile effects of NADPH in the mouse aorta. Rather, NADPH acts as an agonist at two P2X receptor populations; one located on the endothelium and the other on smooth muscle layer.