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

      research-article

      , , *

      Redox Biology

      Elsevier

      NADPH oxidase, Cholesterol, Cell-free system, Arachidonic acid activation, Superoxide production, AA, arachidonic acid, PBS, phosphate buffer saline, Cyt b558, cytochrome b558, Cytc, cytochrome c, DTT, dithiotreitol, EDTA, ethylenediaminetetraacetic acid, FAD, flavin adenine dinucleotide, FMLP, formyl-methionyl-leucyl-phenylalanine, GTP, guanosine-5′-triphosphate, HEPES, [4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid, IPTG, isopropylthiogalatoside, LB, Luria Bertoni, LDL, low density lipoprotein, LR, lipid raft, MF, membrane fractions, MβCD, methyl-β-cyclodextrin, NADPH, reduced β-nicotinamide adenine dinucleotide phosphate, PMSF, phenylmethanesulfonyl fluoride, PtdIns(3)P, phosphatidyl-inositol3-phosphate, PtdIns(3,4)P2, phosphatidylinositol(3,4)-bisphosphate, PX, phox homology domain, ROS, reactive oxygen species, SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

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          Abstract

          The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O 2 •−, 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. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O 2 •−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.

          Highlights

          • Natural cholesterol is important for the NADPH oxidase function.

          • Added cholesterol alone activates slightly the NADPH oxidase.

          • Cholesterol addition lowers the AA dependent activity of NADPH oxidase.

          • Added cholesterol acts on both cytosolic and membrane components.

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          Most cited references 76

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          A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases.

          The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H2O2. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The reaction stoichiometry of Amplex Red and H2O2 was determined to be 1:1. This probe allows detection of 5 pmol H2O2 in a 96-well fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2 release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed assay using Amplex Red results in an increase in fluorescence on oxidation rather than a decrease in fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H2O2 in a variety of oxidase-mediated reactions and very low levels of H2O2 in food, environmental waters, and consumer products. Copyright 1997 Academic Press.
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            Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.

            The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.
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              How human neutrophils kill and degrade microbes: an integrated view.

              Neutrophils constitute the dominant cell in the circulation that mediates the earliest innate immune human responses to infection. The morbidity and mortality from infection rise dramatically in patients with quantitative or qualitative neutrophil defects, providing clinical confirmation of the important role of normal neutrophils for human health. Neutrophil-dependent anti-microbial activity against ingested microbes represents the collaboration of multiple agents, including those prefabricated during granulocyte development in the bone marrow and those generated de novo following neutrophil activation. Furthermore, neutrophils cooperate with extracellular agents as well as other immune cells to optimally kill and degrade invading microbes. This brief review focuses attention on two examples of the integrated nature of neutrophil-mediated anti-microbial action within the phagosome. The importance and complexity of myeloperoxidase-mediated events illustrate a collaboration of anti-microbial responses that are endogenous to the neutrophil, whereas the synergy between the phagocyte NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and plasma-derived group IIA phospholipase A(2) exemplifies the collective effects of the neutrophil with an exogenous factor to achieve degradation of ingested staphylococci.
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                Author and article information

                Contributors
                Journal
                Redox Biol
                Redox Biol
                Redox Biology
                Elsevier
                2213-2317
                5 November 2014
                5 November 2014
                2014
                : 3
                : 16-24
                Affiliations
                Laboratoire de chimie physique, UMR 8000, Université Paris Sud-CNRS, Orsay 91405, France
                Author notes
                [* ]Corresponding author. Chantal.houee@ 123456u-psud.fr
                Article
                S2213-2317(14)00107-4
                10.1016/j.redox.2014.10.001
                4221629
                © 2014 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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