Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.

Degradation pathway of bisphenol A: Does ipso substitution apply to phenols containing a quaternary alpha-carbon structure in the para position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carboncarbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary alpha-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary alpha-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type 11 ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl) phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an 18 02 atmosphere were performed. One atom of 180, was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary a-carbon.

Aldose reductase and the importance of experimental design

Kinetic studies on the AR (aldose reductase) protein have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. As with non-enzymatic glycation reactions, there is probably a free radical element involved derived from monosaccharide autoxidation. in the case of AR, there is free radical oxidation of NADPH by autoxidizing monosaccharides, which is enhanced in the presence of the NADPH-binding protein. Thus any assay for AR based on the oxidation of NADPH in the presence of autoxidizing monosaccharides is invalid, and tissue AR measurements based on this method are also invalid, and should be reassessed. AR exhibits broad specificity for both hydrophilic and hydrophobic aldehydes that suggests that the protein may be involved in detoxification. The last thing we would want to do is to inhibit it. ARIs (AR inhibitors) have a number of actions in the cell which are not specific, and which do not involve them binding to AR. These include peroxy-radical scavenging and effects of metal ion chelation. The AR/ARI story emphasizes the importance of correct experimental design in all biocatalytic experiments. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has led to the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K-m and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimizes the error in the parameters estimated, and is suitable for simple or complex steady-state models.

Neuroinflammation and its modulation by flavonoids

There is increasing evidence to suggest that neuroinflammatory processes contribute to the cascade of events that lead to the progressive neuronal damage observed in neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. Therefore, treatment regimes aimed at modulating neuroinflammatory processes may act to slow the progression of these debilitating brain disorders. Recently, a group of dietary polyphenols known as flavonoids have been shown to exert neuroprotective effects in vivo and in neuronal cell models. In this review we discuss the evidence relating to the modulation of neuroinflammation by flavonoids. We highlight the evidence which suggests their mechanism of action involves: 1) attenuation of the release of cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α); 2) an inhibitory action against inducible nitric oxide synthase (iNOS) induction and subsequent nitric oxide (NO•) production; 3) inhibition of the activation of NADPH oxidase and subsequent reactive oxygen species generation; 4) a capacity to down-regulate the activity of pro-inflammatory transcription factors such as nuclear factor-κB (NF-κB); and 5) the potential to modulate signalling pathways such as mitogen-activated protein kinase (MAPK) cascade. We also consider the potential of these dietary compounds to represent novel therapeutic agents by considering their metabolism in the body and their ability to access the brain via the blood brain barrier. Finally, we discuss future areas of study which are necessary before dietary flavonoids can be established as therapeutic agents against neuroinflammation.

Fish oil fatty acids improve postprandial vascular reactivity in healthy men

Chronic fish oil intervention had been shown to have a positive impact on endothelial function. Although high-fat meals have often been associated with a loss of postprandial vascular reactivity, studies examining the effects of fish oil fatty acids on vascular function in the postprandial phase are limited. The aim of the present study was to examine the impact of the addition of fish oil fatty acids to a standard test meal on postprandial vascular reactivity. A total of 25 men received in a random order either a placebo oil meal (40 g of mixed fat; fatty acid profile representative of the U.K. diet) or a fish oil meal (31 g of mixed fat and 9 g of fish oil) on two occasions. Vascular reactivity was measured at baseline (0 h) and 4 h after the meal by laser Doppler iontophoresis, and blood samples were taken for the measurement of plasma lipids, total nitrite, glucose and insulin. eNOS (endothelial NO synthase) and NADPH oxidase gene expression were determined in endothelial cells after incubation with TRLs (triacylglycerol-rich lipoproteins) isolated from the plasma samples taken at 4 h. Compared with baseline, sodium nitroprusside (an endothelium-independent vasodilator)-induced reactivity (P = 0.024) and plasma nitrite levels (P = 0.001) were increased after the fish oil meal. In endothelial cells, postprandial TRLs isolated after the fish oil meal increased eNOS and decreased NADPH oxidase gene expression compared with TRLs isolated following the placebo oil meal (P <= 0.03). In conclusion, meal fatty acids appear to be an important determinant of vascular reactivity, with fish oils significantly improving postprandial endothelium-independent vasodilation.

Nox2 regulates endothelial cell cycle arrest and apoptosis via p21 (cip1) and p53

Endothelial cells (EC) express constitutively two major isofonns (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 similar to 1:13), but was upregulated 24 It after starvation and increased to 8 +/- 3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28 +/- 0.18-fold increase in O-2(-); production, a dramatic induction of p21(cip1) and p53, cell cycle arrest, and the onset of apoptosis (all p < 0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8 +/- 0.5fold increase in Nox4 mRNA expression after 36 h of starvation (p < 0.01), neither production nor the p21(cip1) or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O-2(-), through the modulation of p21(cip1) and p53 expression, participates in endothelial cell cycle regulation and apoptosis. (c) 2007 Elsevier Inc. All rights reserved.

Absorption and metabolism of olive oil secoiridoids in the small intestine

The secoiridoids 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA) account for approximately 55 % of the phenolic content of olive oil and may be partly responsible for its reported human health benefits. We have investigated the absorption and metabolism of these secoiridoids in the upper gastrointestinal tract. Both 3,4-DHPEA-EDA and 3,4-DHPEA-EA were relatively stable under gastric conditions, only undergoing limited hydrolysis. Both secoiridoids were transferred across a human cellular model of the small intestine (Caco-2 cells). However, no glucuronide conjugation was observed for either secoiridoid during transfer, although some hydroxytyrosol and homovanillic alcohol were formed. As Caco-2 cells are known to express only limited metabolic activity, we also investigated the absorption and metabolism of secoiridoids in isolated, perfused segments of the jejunum and ileum. Here, both secoiridoids underwent extensive metabolism, most notably a two-electron reduction and glucuronidation during the transfer across both the ileum and jejunum. Unlike Caco-2 cells, the intact small-intestinal segments contain NADPH-dependent aldo-keto reductases, which reduce the aldehyde carbonyl group of 3,4-DHPEA-EA and one of the two aldeydic carbonyl groups present on 3,4-DHPEA-EDA. These reduced forms are then glucuronidated and represent the major in vivo small-intestinal metabolites of the secoiridoids. In agreement with the cell studies, perfusion of the jejunum and ileum also yielded hydroxytyrosol and homovanillic alcohol and their respective glucuronides. We suggest that the reduced and glucuronidated forms represent novel physiological metabolites of the secoiridoids that should be pursued in vivo and investigated for their biological activity.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca2+ - and K+ -permeable conductance in root cells

Plant cell growth and stress signaling require Ca2+ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH_. In root cells, extracellular OH_ activates a plasma membrane Ca2+-permeable conductance that permits Ca2+ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca2+-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH_-activated Ca2+- and K+-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca2+ in response to OH_. An OH_-activated Ca2+ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca2+-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca2+ in plants.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Orange juice–derived flavanone and phenolic metabolites do not acutely affect cardiovascular risk biomarkers: a randomized, placebo-controlled, crossover trial in men at moderate risk of cardiovascular disease

Background: Epidemiological data suggest inverse associations between citrus flavanone intake and cardiovascular disease (CVD) risk. However, insufficient randomized controlled trial (RCT) data limit our understanding of mechanisms by which flavanones and their metabolites potentially reduce cardiovascular (CV) risk factors. Objective: We examined the effects of orange juice or a dose-matched hesperidin supplement on plasma concentrations of established and novel flavanone metabolites and their effects on CV risk biomarkers in men at moderate CVD risk. Methods: In an acute, randomized, placebo-controlled crossover trial, 16 fasted participants (aged 51-69 y) received orange juice or a hesperidin supplement (both providing 320 mg hesperidin) or control (all matched for sugar and vitamin C content). At baseline and 5 h post-intake, endothelial function (primary outcome), further CV risk biomarkers (i.e. blood pressure, arterial stiffness, cardiac autonomic function, platelet activation and NADPH oxidase gene expression) and plasma flavanone metabolites were assessed. Prior to each intervention, a diet low in flavonoids, nitrate/nitrite, alcohol and caffeine was followed and a standardized low-flavonoid evening meal was consumed. Results: Orange juice intake significantly elevated mean (± SEM) plasma concentrations of 8 flavanone (1.75 ± 0.35 µmol/L, P < 0.0001) and 15 phenolic metabolites (13.27 ± 2.22 µmol/L, P < 0.0001) compared with control at 5 h post-consumption. Despite increased plasma flavanone and phenolic metabolite concentrations, CV risk biomarkers were unaltered. Following hesperidin supplement intake, flavanone metabolites were not different to control, suggesting altered absorption/metabolism compared with the orange juice matrix. Conclusions: Following single-dose flavanone intake within orange juice, we detected circulating flavanone and phenolic metabolites collectively reaching a concentration of 15.20 ± 2.15 µmol/L but observed no effect on CV risk biomarkers. Longer-duration RCTs are required to further examine the previous associations between higher flavanone intakes and improved cardiovascular health and to ascertain the relative importance of food matrix and flavanone-derived phenolic metabolites.

p22phox C242T SNP inhibits inflammatory oxidative damage to endothelial cells and vessels

Background— T NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism (SNP) C242T of the p22phox subunit of NADPH oxidase has been reported to be negatively associated with coronary heart disease (CHD) and may predict disease prevalence. However, the underlying mechanisms remain unknown. Methods and Results— Using computer molecular modelling we discovered that C242T SNP causes significant structural changes in the extracellular loop of p22phox and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22phox reduced significantly Nox2 expression but had no significant effect on basal endothelial O2.- production or the expression of Nox1 and Nox4. When cells were stimulated with TNFα (or high glucose), C242T p22phox inhibited significantly TNFα-induced Nox2 maturation, O2.- production, MAPK and NFκB activation and inflammation (all p<0.05). These C242T effects were further confirmed using p22phox shRNA engineered HeLa cells and Nox2-/- coronary microvascular endothelial cells. Clinical significance was investigated using saphenous vein segments from non CHD subjects after phlebectomies. TT (C242T) allele was common (prevalence of ~22%) and compared to CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2.- generation in response to high glucose challenge. Conclusions— C242T SNP causes p22phox structural changes that inhibit endothelial Nox2 activation and oxidative response to TNFα or high glucose stimulation. C242T SNP may represent a natural protective mechanism against inflammatory cardiovascular diseases.