Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages.

Thioxo-dihydroquinazolin-one Compounds as Novel Inhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is a key antimicrobial enzyme, playing a normal role in host defense, but also contributing to inflammatory conditions including neuroinflammatory diseases such as Parkinson's and Alzheimer's. We synthesized and characterized more than 50 quinazolin-4(1H)-one derivatives and showed that this class of compounds inhibits MPO with IC50 values as low as 100 nM. Representative compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not result in the accumulation of MPO Compound II.

Nox4: a hydrogen peroxide-generating oxygen sensor.

Nox4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for Nox4 require upstream activators, either calcium or organizer/activator subunits (p47(phox), NOXO1/p67(phox), and NOXA1). Nox4 may also be unusual as it reportedly releases hydrogen peroxide (H₂O₂) in contrast to Nox1-Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection.

High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases.

Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors.

Amyloid-β and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.

Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5-30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors).

NOX2 As a Target for Drug Development: Indications, Possible Complications, and Progress.

Significance: NOX2 is important for host defense, and yet is implicated in a large number of diseases in which inflammation plays a role in pathogenesis. These include acute and chronic lung inflammatory diseases, stroke, traumatic brain injury, and neurodegenerative diseases, including Alzheimer's and Parkinson's Diseases.

Recent Advances: Recent drug development programs have targeted several NOX isoforms that are implicated in a variety of diseases.

Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1.

The mammalian gut microbiota is essential for normal intestinal development, renewal, and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells.

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species.

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems.

On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation.

L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH oxidase (Nox)-derived superoxide (O2(*-)) and identify Nox inhibitors. Yet understanding of the free radical chemistry of the L-012 probe is still lacking. We report that peroxidase and H2O2 induce superoxide dismutase (SOD)-sensitive, L-012-derived CL in the presence of oxygen.

Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited.

Reactive oxygen species (ROS) are a chemical class of molecules that have generally been conceptualized as deleterious entities, albeit ones whose destructive properties could be harnessed as antimicrobial effector functions to benefit the whole organism. This appealingly simplistic notion has been turned on its head in recent years with the discovery of the NADPH oxidases, or Noxes, a family of enzymes dedicated to the production of ROS in a variety of cells and tissues. The Nox-dependent, physiological generation of ROS is highly conserved across virtually all multicellular life, often as a generalized response to microbes and/or other exogenous stressors.

Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair.

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1.

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

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.

Microbicidal activity of vascular peroxidase 1 in human plasma via generation of hypochlorous acid.

Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections.

Congenital heart disease affects cerebral size but not brain growth.

Infants with congenital heart disease (CHD) have delayed brain maturation and alterations in brain volume. Brain metrics is a simple measurement technique that can be used to evaluate brain growth. This study used brain metrics to test the hypothesis that alterations in brain size persist at 3 months of age and that infants with CHD have slower rates of brain growth than control infants.

Regional alterations in cerebral growth exist preoperatively in infants with congenital heart disease.

Objectives: Magnetic resonance imaging has been used to define the neurologic abnormalities in infants with congenital heart disease (CHD), including preoperative injury and delayed brain maturation. The present study used qualitative scoring, cerebral biometry, and diffusion imaging to characterize the preoperative brain abnormalities in infants with CHD, including the identification of regions of greater vulnerability.

Methods: A total of 67 infants with CHD had preoperative magnetic resonance imaging scans available for analysis of brain injury using qualitative scoring and brain development using qualitative scoring, metrics, and diffusion imaging.

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4.

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-).

Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain.

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits.

Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals.

The production of reactive oxygen species (ROS) in a highly regulated fashion is a hallmark of members of the NADPH oxidase (Nox) family of enzymes. Nox enzymes are present in most eukaryotic groups such as the amebozoid, fungi, algae and plants, and animals, in which they are involved in seemingly diverse biological processes. However, a comprehensive survey of Nox functions throughout biology reveals common functional themes.

Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling.

Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function, transgenic mice overexpressing Nox1 in smooth muscle cells (Tg(SMCnox1)) were subjected to angiotensin II (ANG II)-induced hypertension. As expected, NADPH-dependent superoxide generation was increased in aortas from Nox1-overexpressing mice.

Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.

Unlabelled: Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4).

Oxidative modification of nuclear mitogen-activated protein kinase phosphatase 1 is involved in transforming growth factor beta1-induced expression of plasminogen activator inhibitor 1 in fibroblasts.

Transforming growth factor beta (TGF-beta) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-beta. The molecular mechanisms whereby TGF-beta increases ROS production and ROS modulate the signaling processes of TGF-beta, however, remain poorly defined. In this study, we show that TGF-beta1 stimulates NADPH oxidase 4 (Nox4) expression and ROS generation in the nucleus of murine embryo fibroblasts (NIH3T3 cells).

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

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.