The effect of and its postbiotics on the expression of genes involved in the endocannabinoid system and intestinal epithelial integrity in Caco-2 cells.

Purpose: Gut microbiota and its derivatives by constantly interacting with the host, regulate the host function. Intestinal epithelium integrity is under the control of various factors including the endocannabinoid system (ECS). Accordingly, we aimed at investigating the effect of and its postbiotics (i.

Role of the phospholipid binding sites, PX of p47 and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2.

In phagocytes, superoxide anion (O), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b (cyt b) and four cytosolic components: p40, p47, p67, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways.

New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393 cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation.

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 and p22) forming the catalytic core, three cytosolic proteins (p67, p47 and p40) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components.

Accumulation of Cytochrome at the Plasma Membrane: Hallmark of Oxidative Stress in Phagocytic Cells.

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.

Radiation-induced reactive oxygen species partially assemble neutrophil NADPH oxidase.

Neutrophils are key cells from the innate immune system that destroy invading bacteria or viruses, thanks mainly to the non-mitochondrial reactive oxygen species (ROS) generated by the enzyme NADPH oxidase. Our aim was to study the response of neutrophils to situations of oxidative stress with emphasis on the impact on the NADPH oxidase complex. To mimic oxidative stress, we used gamma irradiation that generated ROS (OH, O and HO) in a quantitative controlled manner.

Impacts of vesicular environment on Nox2 activity measurements in vitro.

Background: The production of superoxide anions (O) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate.

A Close-Up View of the Impact of Arachidonic Acid on the Phagocyte NADPH Oxidase.

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear.

Phagocyte NADPH oxidase, oxidative stress and lipids: Anti- or pro ageing?

The role of NADPH oxidase in ageing is debated because of the dual roles of free radicals, toxic though necessary. In this paper we summarize some results about two aspects linked to the regulation of the activity of phagocyte NADPH oxidase (Nox2), encountered frequently in elderly people: inflammation and hypercholesterolemia. In the presence of a high amount of reactive oxygen species (ROS) created by itself or by any other source, the enzyme activity is mostly lowered.

Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits.

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22) and four cytosolic proteins (p40, p47, p67 and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane.

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

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 (Cytb ), constituted by the tight association of the gp91 (also named Nox2) and p22 proteins.

The physicochemical properties of membranes correlate with the NADPH oxidase activity.

Background: Phagocytes kill ingested microbes by exposure to high concentrations of toxic reactive species generated by NADPH-oxidases. This membrane-bound electron-transferring enzyme is tightly regulated by cellular signaling cascades. So far, molecular and biophysical studies of the NADPH-oxidase were performed over limited temperature ranges, which weaken our understanding of immune response or inflammatory events.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase.

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex.

Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study.

The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2(•-), which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol.

Assembly of phagocyte NADPH oxidase: A concerted binding process?

Background: The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67(phox), p47(phox), p40(phox) and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.

Recombinant form of mammalian gp91(phox) is active in the absence of p22(phox).

The flavocytochrome b558 of the phagocyte NADPH oxidase complex comprises two membrane proteins, a glycosylated gp91phox and a non-glycosylated p22phox. Gp91phox contains all of the redox carriers necessary to reduce molecular oxygen to superoxide using NADPH. The capacity of gp91phox to produce superoxide in the absence of its membrane partner p22phox has been little studied.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components.

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox).

Modulation of the activity of the NADPH oxidase system by reactive oxygen species: influence of catalase.

The nicotinamide adenine dinucleotide phosphate oxidase complex (Nox) is a major source of non-mitochondrial reactive oxygen species in cells. Nox contains both membrane (Cytb(558)) and cytosolic (p40(phox), p47(phox), p67(phox) and Rac) components. Nox has been submitted to a combination of oxygen free radicals produced by irradiation and to hydrogen peroxide.

Targeting NADPH-oxidase by reactive oxygen species reveals an initial sensitive step in the assembly process.

NADPH-oxidase (Nox) is a highly regulated dynamic complex comprising membrane and cytosolic proteins and is the major source of nonmitochondrial cellular reactive oxygen species (ROS). In phagocyte cells, in which ROS are produced in huge amounts, Nox is "naturally" assailed by the action of its own ROS. We have subjected each individual component of Nox or the whole complex at various times during the assembly process either to oxygen free radicals produced by radiolysis or to hydrogen peroxide.

Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system.

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b(558) (Cytb(558)) which consists of a heterodimer of the gp91(phox) and p22(phox) subunits. The Cytb(558) is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb(558) (rCytb(558)).

Are the fluorescent properties of the cyan fluorescent protein sensitive to conditions of oxidative stress?

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative gamma-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the OH radical, the protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra.

The cytosolic subunit p67phox of the NADPH-oxidase complex does not bind NADPH.

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558.

Preliminary electrochemical studies of the flavohaemoprotein from Ralstonia eutropha entrapped in a film of methyl cellulose: Activation of the reduction of dioxygen.

A flavohaemoprotein (FHP) from Ralstonia eutropha, obtained in a pure and active form, has been entrapped in a film of methyl cellulose on the electrode surface and gives a stable and reproducible electrochemical response at pH 7.00 when subject to cyclic voltammetry using a glassy carbon electrode. To our knowledge, no previous direct electrochemistry had been achieved with a bacterial flavohaemoglobin, which possess both a FAD and a haem.